Based on a comparison of the dominant microbial populations in 17 pig manure samples and using a molecular typing method, we identified a species, Lactobacillus sobrius and Lactobacillus amylovorus (which now are considered a single species and are designated L. sobrius/amylovorus here), that was consistently found in manure. The aim of the present study was to confirm by real-time PCR the relevance of this species as a marker of pig fecal contamination. The specificity of L. sobrius/amylovorus was evaluated in human and animal DNA extracted from feces. The real-time PCR assay then was applied to water samples, including effluents from urban wastewater treatment plants, runoff water, and rivers. L. sobrius/amylovorus was consistently present in all samples of swine origin: 48 fecal samples, 18 from raw manure and 10 from biologically treated manure at mean concentrations of 7.2, 5.9, and 5.0 log10 cells/g, respectively. The species was not detected in any of the other livestock feces (38 samples from cattle and 16 from sheep), in the 27 human fecal samples, or in the 13 effluent samples from urban wastewater treatment plants. Finally, L. sobrius/amylovorus was not detected in runoff water contaminated by cattle slurry, but it was quantified at concentrations ranging from 3.7 to 6.5 log10 cells/100 ml in runoff water collected after pig manure was spread on soil. Among the stream water samples in which cultured Escherichia coli was detected, 23% tested positive for L. sobrius/amylovorus. The results of this study indicate that the quantification of L. sobrius/amylovorus using real-time PCR will be useful for identifying pig fecal contamination in surface waters.