In December 2003, forensic investigators extracted enough material from a mixed blood sample found on JonBenét's underwear to establish a DNA profile.[7] The DNA belongs to an unknown male. The DNA was submitted to the FBI's Combined DNA Index System (CODIS), a database containing more than 1.6 million DNA profiles, mainly from convicted felons. The sample has yet to find a match in the database, although it continues to be checked for partial matches on a weekly basis.
Later investigations also discovered that there were more than 100 burglaries in the Ramseys' neighborhood in the months before JonBenét's murder, and that 38 registered sex offenders were living within a two-mile (3 km) radius of the Ramseys' home - an area that encompasses half the population of the city of Boulder - but that none of the sex offenders had any involvement in the murder.
On August 16, 2006, 41-year-old John Mark Karr, a former schoolteacher, confessed to the murder while being held on child pornography charges from Sonoma County, California. Authorities reportedly tracked him down using the Internet after he sent e-mails regarding the Ramsey case to Michael Tracey, a journalism professor at the University of Colorado.[9] Once apprehended, he confessed to being with JonBenét when she died, stating that her death was an accident. When asked if he was innocent, he responded, "No."
However, Karr's DNA did not match that found on JonBenét Ramsey's body. On August 28, 2006, prosecutors announced that no charges would be filed against him for the murder of JonBenét Ramsey. In early December 2006, Department of Homeland Security officials reported that federal investigators were continuing to explore whether Karr had been a possible accomplice in the killing.
No evidence has ever come to light that placed the then-married Alabama resident Karr near Boulder during the Christmas 1996 crime. Evidence linking Karr to the killing is highly circumstantial in nature. For instance, handwriting samples taken from Karr were said to match the ransom note. In particular, his technique for writing the letters E, T and M were described by the media as being very rare.

The bristle end of the paint brush was found in a tub of Patsy Ramsey's art supplies, but the bottom third was never located despite extensive searching of the house by law enforcement in subsequent days. Experts noted that the construction of the garrote required a special knowledge of knots. Autopsy also revealed that the child had eaten pineapple only a few hours before the murder. Photographs of the home, taken the day JonBenét's body was found, show a bowl of pineapple on the kitchen table with a spoon in it, and police reported finding Patsy's fingerprints on it. However, neither Patsy nor John claim to remember putting this bowl on the table or feeding pineapple to JonBenét.
While it was reported that no footprints led to the house on snow-covered ground, other reporters found that snow around the doors of the house was cleared away. Police reported no signs of forced entry, although a basement window that had been broken and left unsecured before Christmas, along with other open doors, were not reported to the public until a year later.

In the afternoon of the same day, Boulder Police Detective Linda Arndt asked Fleet White, a friend of the Ramseys, to take John Ramsey and search the house for "anything unusual." John Ramsey and two of his friends started their search in the basement. After first searching the bathroom and "train room," the two went to a "wine cellar" room where Ramsey found his daughter's body covered in a white blanket.
The police were later claimed by observers to have made several critical mistakes in the investigation, such as not sealing off the crime scene and allowing friends and family in and out of the house once a kidnapping was reported.
Critics of the investigation have since claimed that officers also did not sufficiently attempt to gather forensic evidence before or after JonBenet's body was found, possibly because they immediately suspected the Ramseys in the killing. Some officers holding these suspicions reported them to local media, who began reporting on January 1 that the assistant district attorney thought "it's not adding up"; the fact that the body of the girl was found in her own home was considered highly suspicious by the investigating officers. The results of the autopsy revealed that JonBenét was killed by strangulation and a skull fracture. A garrote made from a length of tweed cord and the broken handle of a paintbrush had been used to strangle her; her skull had suffered severe blunt trauma; there was no evidence of conventional rape, although sexual assault could not be ruled out. The official cause of death was asphyxiation due to strangulation associated with craniocerebral trauma.