A new mayfly species, Heptagenia whitingi Webb & McCafferty n.sp. is described from larvae, a male subimago, a female adult, and eggs collected from large rivers in the west-central portion of North America. Larvae are differentiated from other North American Heptagenia Walsh by a pair of large, rectangular pale markings on abdominal tergum 4, and the combination of having the posterior margin of the abdominal terga with bluntly pointed spines less than half the length of the fine setae, small blunt spines on the posterior margin of the caudal filaments, and numerous rows of setae laterally on the ventral surface of the labrum. A 630 bp partial sequence of the mitochondrial gene cytochrome oxidase 1 (COI) from three specimens of H. whitingi n.sp. was compared with those of 12 specimens representing eight other North American species of Heptagenia. Intraspecific sequence divergences based on Kimura-2-parameter (K2P) distance ranged from 0–1.1%. Interspecific sequence divergence based on K2P distance ranged from 8.9–20.0%. Heptagenia whitingi n.sp. differed from its sister taxon H. flavescens (Walsh) by 11.7%. Heptagenia diabasia Burks and H. elegantula (Eaton) differed from each other by only 1.1%; these two alleged species show a clinal pattern in larval abdominal coloration and there are no structural differences between the semaphorants. On this basis, H. diabasia is placed as a junior subjective synonym of H. elegantula, n.syn.