Group B Streptococcus (GBS) is a leading cause of infant sepsis and meningitis, causing significant morbidity and mortality worldwide.1,2,3 GBS infections within the first week of life are classified as early-onset disease (EOD), while those arising between one week and three months are late-onset disease (LOD)1. While patient characteristics may affect the clinical presentation of invasive GBS disease, bacterial genetic factors could also contribute. Genome-wide association studies (GWAS) are a powerful tool to this end, but they face challenges in bacterial populations due to high clonality and unique genetic components like insertions/deletions and pan-genome components4. We developed a novel, publicly accessible Nextflow pipeline for GBS whole-genome assembly and GWAS, suited for bacterial genome complexity.5 It performs single nucleotide polymorphism (SNP) GWAS to test the association of core genome SNPs, PanGWAS to test the association of accessory genes, and De Brujin Graph GWAS to test the association of unitigs, a useful unit at the intersection of core and accessory genome variation. The pipeline can analyze binary or continuous phenotypes and addresses high clonality using various population structure correction methods, balancing computational intensity and statistical power. We validated the pipeline on 500 publicly available GBS genome sequences from neonatal GBS cases deposited by the US CDC, aiming to identify genetic factors related to age of disease onset classified into EOD and LOD. This pipeline serves as a framework for other bacterial pathogens. This pipeline can be a useful, accessible tool for the scientific community to further explore the genomic epidemiology of GBS