Objective - To compare the sensitivity of polymerase chain reaction (PCR) with microbiological culture for detecting salmonellae in equine fecal samples and equine environmental swab specimens. Design - Samples and specimens were tested by PCR and microbiological culture. Sample Population - A fecal sample from each of 152 horses admitted consecutively to the clinic for evaluation by the outpatient service, 282 fecal samples from 110 hospitalized horses that had been submitted to the clinical microbiology laboratory, and 313 environmental swab specimens were examined. Procedure - Each sample and specimen in the study was tested, using PCR and microbiological culture. Results of PCR and culture were compared. Results - Significantly (P < 0.001) more fecal samples were positive by PCR than by microbiological culture. 26 of 152 (17.1%) fecal samples collected from horses admitted by the outpatient service were positive by PCR and none was positive by culture. 71 of 110 hospitalized horses were identified as positive by PCR, compared with 11 horses identified as positive by culture. All culture-positive horses were positive by PCR. Of the 11 culture-positive horses, 10 (90.9%) were identified as PCR positive after testing of the first sample submitted, compared with 7 (63.6%) by culture. All PCR-positive horses were detected after a total of 3 samples/horse were submitted, whereas as many as 5 samples/horse was required to identify all culture-positive horses. 8 of 313 environmental specimens were positive by PCR, and none was positive by culture. Conclusion - The PCR method reported here was more sensitive, more rapid, and required submission of fewer samples or specimens than did microbiological culture for detecting salmonellae.