Streptococcus (S.) agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, as well as various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data is needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from different sources and geographical origins. To this end, 373 S. agalactiae isolates from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different strains were identified. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. The findings of our study suggest that while S. agalactiae is well adapted to various hosts (including humans, cattle, dogs and other species), interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The presented virulence and resistance gene profiles enable new insights into interspecies transmission and make a crucial contribution in the identification of suitable targets for therapeutic agents and vaccines.