Aims The aim of this study was to develop a TaqMan quantitative polymerase chain reaction (qPCR), based on the Streptococcus agalactiae groEL gene, to specifically quantify levels of bacteria within samples derived from aquatic sources, particularly aquaculture. Enumeration of bacteria by qPCR was compared with culture-based methods. Methods and Results The qPCR was sensitive to 33 isolates of S. agalactiae, representing 11 clonal complexes from aquatic, bovine and human hosts. The specificity of the assay was 92 center dot 5% at a threshold C-q value of 35. No cross-reaction with Streptococcus iniae was noted and of the 22 comparator species screened to test assay specificity, Streptococcus porcinus had a C-q value of 33 center dot 7 S, while Streptococcus gallolyticus subsp. macedonicus and Streptococcus ictaluri had one replicate value above the C-q threshold of 35 (34 center dot 5 and 34 center dot 4 respectively), while only S. agalactiae were detected with a C-q value of 30. The limit of detection of the assay was 1 center dot 7 copies per mu l at C-q 35. Discrepancies between molecular and culture-based methods of enumeration were noted. Conclusions The qPCR was able to detect a diverse range of S. agalactiae isolates from different clonal complexes (CCs) and could distinguish between S. agalactiae and closely related species, notably S. iniae. The results suggest that a C-q 30 would be a very meaningful cut-off, allowing the detection of infected fish while ruling out all false positives. Significance and Impact of the Study This rapid and sensitive qPCR assay is useful to quantify DNA copy number in the laboratory and could prove useful for detecting low levels of S. agalactiae in aquaculture systems, including Oreochromis niloticus culture.