To compare the Affirm VPIII molecular test (Becton Dickinson, Burlington, NC) with morphologic identification used in routine Papanicolaou (Pap) test screening in the detection and identification of Candida species, Trichomonas vaginalis, and Gardnerella vaginalis, we identified 431 cases with a concomitant Pap test and Affirm VPIII assay performed from the archives of a large academic institution. The study population consisted of women ranging in age from 17 to 79 years (mean and median ages, 33 and 31 years, respectively). With a routine Pap test, 60 patients (13.9%) were found to have bacterial vaginosis, 60 (13.9%) candidiasis, and 3 (0.7%) Trichomonas infection. With the Affirm VPIII assay, 183 (42.5%) patients tested positive for G vaginalis, 70 (16.2%) positive for Candida species, and 10 (2.3%) positive for T vaginalis. The differences were statistically significant. The results demonstrate that our patient population had a high incidence of bacterial vaginosis/Candida vaginitis; however, the Affirm VPIII was a more sensitive diagnostic test for the detection and identification of all 3 organisms compared with the Pap test.