Fusarium isolates that form part of the Gibberella fujikuroi species complex have been classified using either a morphological, biological, or phylogenetic species concept. Problems with the taxonomy of Fusarium species in this complex are mostly experienced when the morphological and biological species concepts are applied. The most consistent identifications are obtained with the phylogenetic species concept. Results from recent studies have presented an example of discordance between the biological and phylogenetic species concepts, where a group of F. subglutinans sensu stricto isolates, i.e., isolates belonging to mating population E of the G. fujikuroi complex, could be sub-divided into more than one phylogenetic lineage. The aim of this study was to determine whether this sub-division represented species divergence or intraspecific diversity in F. subglutinans. For this purpose, we included 29 F. subglutinans isolates belonging to the E-mating population that were collected from either maize or teosinte, from a wide geographic range. DNA sequence data for six nuclear regions in each of these isolates were obtained and used in phylogenetic concordance analyses. These analyses revealed the presence of two major groups representing cryptic species in F. subglutinans. These cryptic species were further sub-divided into a number of smaller groups that appear to be reproductively isolated in nature. This suggests not only that the existing F. subglutinans populations are in the process of divergence, but also that each of the resulting lineages are undergoing separation into distinct taxa. These divergences did not appear to be linked to geographic origin, host, or phenotypic characters such as morphology.