Aims: To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated by culturing and quantitative polymerase chain reaction (q-PCR). Methods and Results: Dialysis tubing containing river supplemented with overnight cultures of Bifidobacterium adolescentis (BA) and Bifidobacterium dentium (BD) or urban wastewater were suspended in a river for up to 10 days. At intervals, the contents of each dialysis tube were assayed using q-PCR assays for BA and BD, and selective culture media for FC, SRC, total bifidobacteria (TB), sorbitol-fermenting bifidobacteria (SFB) and cultivable BA. Mean summer T-90 values were 251 h for SRC, 92 h for FC, 48 h for BA and BD by q-PCR, and 9 h for TB. Conclusions: Bifidobacterium spp. was the population with the lowest persistence, showing seasonal differences in T-90 when measured by culture techniques or by q-PCR. This difference in relative persistence is because of a longer persistence of molecular targets than cultivable cells. Significance and Impact of the Study: The persistence of a viable bifidobacteria cells is shorter, but the longest persistence of molecular targets. This factor could be used for origin the faecal pollution in water for the development of microbial source tracking (MST).