The Type VI Secretion System (T6SS) is an antagonistic weapon used by Bacteroides spp. to colonise the gut via interbacterial competition. The T6SS genomic locus contains variable regions encoding for diverse toxic effector and cognate immunity proteins, yet how these vary across strains, affect competitive ability and evolve is understudied. We bioinformatically explored T6SS variation in Bacteroides fragilis; we screened B. fragilis genomes for a T6SS locus and grouped loci into variants using sequence similarity clustering. We show that the T6SS is both prevalent and diverse in B. fragilis, with diversity primarily existing in the presence, number and combinations of putative effector and cognate immunity proteins. We also identify a putative mechanism for the evolution of novel effector-immunity pairs within the identified T6SS variants and hypothesise that this mechanism contributes to the emergence of novel strains with differential competitive abilities. To test this hypothesis, we generated in vitro variants of effector-immunity pairs and test the capacity of evolved effector/immunity proteins to mediate both attack and defence. Here, we show that newly evolved proteins have defence phenotypes dissimilar to their parental ones, suggesting that our genomic study captures intermediate evolutionary pathways within the T6SS which might contribute towards the rise of novel dominant strains within the gut microbiome. This study provides a large-scale analysis of T6SS variability in B. fragilis, providing a foundation to study how both the evolution and subsequent variation of the T6SS impacts competition and colonisation of strains within microbial communities.