Previous studies of resistance gene ecology have focused primarily on populations such as hospital patients and farm animals that are regularly exposed to antibiotics. Also, these studies have tended to focus on numerically minor populations such as enterics or enterococci. We report here a cultivation-independent approach that allowed us to assess the presence of antibiotic resistance genes in the numerically predominant populations of the vaginal microbiota of two populations of primates that are seldom or never exposed to antibiotics: baboons and mangabeys. Most of these animals were part of a captive colony in Texas that is used for scientific studies of female physiology and physical anthropology topics. Samples from some wild baboons were also tested. Vaginal swab samples, obtained in connection with a study designed to define the normal microbiota of the female vaginal canal, were tested for the presence of two types of antibiotic resistance genes: tetracycline resistance (tet) genes and erythromycin resistance (erm) genes. These genes are frequently found in human isolates of the two types of bacteria that were a substantial part of the normal microbiota of primates (Firmicutes and Bacteroidetes). Since cultivation was not feasible, polymerase chain reaction and DNA sequencing were used to detect and characterize these resistance genes. The tet(M) and tet(W) genes were found most commonly, and the tet(Q) gene was found in over a third of the samples from baboons. The ermB and ermF genes were found only in a minority of the samples. The ermG gene was not found in any of the specimens tested. Polymerase chain reaction analysis showed that at least some tet(M) and tet(Q) genes were genetically linked to DNA from known conjugative transposons (CTns), Tn916 and CTnDOT. Our results raise questions about the extent to which extensive exposure to antibiotics is the only pressure necessary to maintain resistance genes in natural settings.