Cronobacter (Enterobacter sakazakii) is a recently defined genus consisting of 6 species. To extend our understanding of the genetic relationship between Cronobacter sakazakii BAA-894 and the other species of this genus, microarray-based comparative genomic indexing (CGI) was undertaken to determine the presence/absence of genes identified in the former sequenced genome and to compare 276 selected open reading frames within the different Cronobacter strains. Seventy-eight Cronobacter strains (60 C. sakazakii, 8 C. malonaticus, 5 C. dublinensis, 2 C. muytjensii, 1 C. turicensis, 1 C. genomospecies 1, and 1 Cronobacter sp.) representing clinical and environmental isolates from various geographical locations were investigated. Hierarchical clustering of the CGI data showed that the species grouped as clusters. The 5 C. dublinensis and 2 C. muytjensii strains examined formed distinct species clusters. Moreover, all of the C. sakazakii and 3 of 8 C. malonaticus strains formed a large cluster. The remaining C. malonaticus strains formed a sub-group within a larger cluster that also contained C. turicensis, C. genomospecies 1, and an unknown Cronobacter sp. Cronobacter sakazakii and 3 of 8 C. malonaticus strains could be distinguished from the others within the collection by the presence of 10 fimbrial related genes. Similarly, capsule and/or lipopolysaccharide (LPS) related glycosyltransferases differentiated several of the C. sakazakii strains from each other.