Abstract Background Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. Results We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. Conclusions We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.