Group B Streptococcus (GBS) causes various infections in adults, including urinary tract infection (UTI) and asymptomatic bacteriuria (ABU). Some bacteria that cause ABU can utilize urine as a substrate for growth, which can promote asymptomatic colonization in the host. An analysis of diverse GBS isolates associated with ABU and UTI for growth in human urine has not been undertaken. Here, we examined a large collection of clinical urinary GBS isolates from individuals with acute UTI (n=62), and ABU with bacteriuria ≥104 c.f.u. ml−1 (n=206) or <104 c.f.u. ml−1 (n=90) for their ability to grow in human urine. Among all 358 GBS isolates analysed, 40 exhibited robust growth in urine in contrast to 25 that were unable to grow and non-culturable after incubation in urine. Growth phenotypes were disproportionately represented among the different groups of isolates, whereby robust growth was significantly more likely to be associated with high-grade ABU versus low-grade ABU or acute UTI (38/40 vs. 11/25; odds ratio 4.6, 95% CI, 1.5–14.8). Growth of bacteria in urine can depend on iron bioavailability, and we therefore performed growth assays using urine supplemented with 2,2-dipyridyl to chelate iron. In contrast to a control strain of ABU Escherichia coli, for which iron limitation significantly attenuated growth, iron sequestration had no significant attenuation effect on the growth of ABU GBS strain 834 in urine. Despite this finding, PCR confirmed the presence of several known growth-associated genes in GBS 834, including fhuD for iron uptake. We conclude that GBS adaptation for growth in human urine is more likely to be associated with high-grade ABU than acute UTI, and for GBS 834, this growth trait is not significantly constrained by conditions of iron sequestration.