Group B streptococcus (GBS) causes pyelonephritis in adults but the mechanisms of infection by which GBS infects the kidneys in vivo are unknown. We investigated GBS infection of the kidneys in mice following experimental challenge via the hematogenous route (transient bacteremia model) or transurethral route (bladder infection and cystitis model). Adult female mice were examined for bacterial dissemination to the kidneys and other organ systems at 24–72 h and tissue samples were assessed for histopathological changes. Comparisons included analysis of different challenge inoculum doses ranging between 107-109 CFU and investigation of several GBS serotypes, including representative strains of serotype V (NEM316), III (BM110, 874391) and Ia (807). Mice with transient, low-level GBS bacteremia routinely developed acute pyelonephritis secondary to high-level kidney infection; infection progressed with high GBS burdens that were sustained in the tissue for days in contrast to bacterial clearance in other organs, including spleen, liver and heart. The histopathological changes of acute pyelonephritis due to GBS were characterized using hematoxylin and eosin, and stains for bacteria, neutrophils, macrophages, mast cells and T lymphocytes; this revealed recruitment of a mixed inflammatory cell population that infiltrated the renal medulla of infected mice in focal areas of discrete micro-abscesses. In contrast, bladder infection leading to cystitis in mice did not result in ascending spread of GBS to the kidneys. We conclude that transient bacteremia, rather than preceding infection of the lower urinary tract, is the predominant condition that leads to GBS kidney infection and subsequent development of acute pyelonephritis.