Classification within Eragrostis, a large genus of ca. 400 species in Poaceae subfamily Chloridoideae, has long been problematic. While spikelet disarticulation characters have been the most common basis of infrageneric classifications, they have not been demonstrated to describe monophyletic groups. An alternative classification system linked to leaf blade anatomy characters is explored using phylogenies based on DNA sequence data from three loci (nuclear GBSSI, plastid rps16, and the plastid trnL-F region) in this study. Eragrostis possesses a great deal of variation in leaf blade anatomy, ranging from NAD-ME-like in structure (subgenus Eragrostis) to PCK-like (subgenus Caesiae), with some intermediates between these extremes. This study reveals that a great deal of variation exists in those species traditionally classified as “intermediate” in leaf blade anatomy. It also indicates that the PCK-like species form a monophyletic group in phylogenetic analyses based on plastid data, GBSSI sequences, and simultaneous analysis of all loci. NAD-ME-like and intermediate species do not form monophyletic groups, but phylogenies based on plastid sequences indicate some evolutionary conservation of the various types of intermediate anatomy. This study also evaluates the leaf anatomy type in allopolyploids. A high proportion of these allopolyploids have intermediate leaf anatomy, potentially reflecting their derivation from progenitors with distinct anatomical types.