Cell line cross-contamination as well as genetic drift during passaging have been acknowledged as widespread problems since the 1960s. Improper cell line identification can invalidate results and, if not discovered, pollute the scientific community's body of knowledge with regard to cancer cell lines, their gene expression, and their drug susceptibilities. Despite the obvious need, validation of cell line identity is not yet widely required, and the problem persists. A highly sensitive polymerase chain reaction (PCR)-based approach and short tandem repeat (STR) profiling were used to examine the prevalence of inter- and intraspecies cell line contamination in a veterinary research setting. First, 60 cell lines from 6 laboratories were tested with multiplex species-specific PCR capable of identifying 6 commonly used species. Of these, 3 were determined to be misidentified by species. Second, to identify intraspecies contamination among canine cancer cell lines, 29 canine lines from 3 different laboratories were analyzed with STR fingerprinting. Using this methodology, 3 canine cell lines were determined to be misidentified or cross-contaminated by other canine cell lines. Finally, genetic drift was observed within 1 cell line obtained from different laboratories. These findings emphasize the importance of cell line validation as a critical component of "good cell culture practice." A database of the STR profiles obtained in the current study has been established for future comparison and validation of canine cell lines by investigators at Colorado State University and other institutions.