In the United States, ~1.4 million sporadic human Salmonella enterica infections occur annually, with an estimated 6% attributable to reptile exposure. Detection of Salmonella in reptiles can be challenging given the limitations among detection methods. We evaluated sampling and detection methods for S. enterica in a cross-sectional study of reptilian patients ( n = 45) over the course of 13 mo. Two sampling methods (cloacal swabs, electrostatic cloth body-feet samples) and 3 detection methods (enriched culture, lateral flow immunoassay [LFI], real-time PCR) were compared using McNemar and Fisher exact tests. Results varied by species, sample type, and detection method. In total, 14 of 45 (33%) patients were positive by culture, 10 of 45 (22%), and/or 13 of 45 (29%) by rtPCR. Among rtPCR-positive results, cloacal swabs (12 of 45 [27%]) resulted in a higher detection than body-feet wipes (4 of 45 [9%]; p = 0.01). Among culture-positive results, shedding was most commonly detected after additional incubation at room temperature when testing cloacal swabs (9 of 45 [20%]). However, there was significant disagreement between sampling methods (cloacal vs. body-feet; p = 0.03). No samples were positive by LFI. In general, cloacal swabs yielded the highest test-positive rates, irrespective of testing method. Our study highlights the importance of using detection methods optimized for the sample being tested.