Aims To develop a pcsB-based Loop-mediated isothermal amplification (LAMP) method for the detection of Streptococcus agalactiae (GBS) in milk, tilapia and vaginal swabs. Methods and Results The sensitivity of the LAMP method using real-time turbidity monitoring was 1 pg of template within 1 h at 64 degrees C, 100-fold higher than conventional PCR. The sensitivity of visual detection dropped an order of magnitude using SYBR Green I or hydroxynaphthol blue. The validity of the visual LAMP assay was assessed by the detection of GBS in 180 vaginal swabs from one hospital, 14 brain tissues samples of diseased tilapias from two fishponds and fresh milk of 67 dairy cattle from one farm. In total, 17 samples (4 vaginal swabs, 13 tilapia brain tissues but no milk sample) tested positive for GBS. Subsequent bacterial identification confirmed the specificity and reliability of the LAMP method. Molecular serotyping and multilocus sequence typing demonstrated that all 13 tilapia GBS isolates were identical (serotype Ia, ST7), whereas the four human GBS isolates were more diverse and could be classified into two serotypes (Ia, III) and four sequence types (ST19, ST23, ST24, ST862). Virulence gene testing showed that only the bac, rib and lmb genes were not present in all isolates. Antimicrobial susceptibility profiles of the isolates were basically consistent with their genotypes, except for sulphonamide and fluoroquinolone. Conclusions We developed a reliable pcsB-based LAMP assay for GBS detection. Our results demonstrated that the prevalence of GBS was 92 center dot 9% among diseased tilapia, 2 center dot 2% among female patients and 0% on a dairy farm in Hainan. Significance and Impact of the Study The pcsB-based LAMP method is suitable for GBS detection and contains great potential of application in dairy industry, aquiculture and clinical.