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To determine presence of bacterial DNA in canine stifles with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL) compared to normal canine stifles (control).Prospective clinical study.Dogs (n = 44).Dogs of varying age, breed, sex, and weight residing in California were assessed for stifle pathology (CCLR, MPL, or normal control). Synovial fluid of all stifles was assessed for the presence of bacterial DNA using broad-ranging 16S rRNA primers and PCR.Bacterial DNA was detected in normal control stifles and those with CCLR and MPL. There were no statistical differences in the copy numbers of bacterial DNA in the stifle synovial fluid among groups (P > .05); however, synovial fluid specimens from dogs with stifle pathology (CCLR and MPL combined) tended to have higher copy numbers of bacterial DNA than those from controls (P = .06). There was no significant difference in the number of bacterial DNA between the CCLR and MPL groups (P = .57). The copy numbers of bacterial DNA had a weak positive significant correlation with the duration of lameness in CCLR group (P < .05).Increased detection of bacterial DNA in the stifle synovial fluid may indicate joint pathology but not be directly linked to a specific joint disease.