Simple Summary Heptageniidae is the third most species-rich family within Ephemeroptera (mayflies). Although multiple studies have been conducted, the monophyly, phylogenetic relationships, and the divergence time of its subfamilies have always been controversial. The current study sequenced 17 new mitogenomes to reconstruct the phylogenetic relationships and calculate the divergence time within Heptageniidae. Therefore, based on comparing the composition of three mitogenomes bias, phylogenetic relationships, and divergence time of Epeorus montanus Brodsky, 1930, we suggest that cryptic species exist in E. montanus.Abstract Heptageniidae are known for their flat heads and bodies and are divided into three subfamilies. Despite the extensive diversity within this group and considerable efforts made to understand their evolutionary history, the internal classifications and origin time of Heptageniidae remains controversial. In this study, we newly sequenced 17 complete mitogenomes of Heptageniidae to reconstruct their phylogenetic positions within this family. Because of the ambiguous time of origin, our study also estimated the divergence time within Heptageniidae based on five fossil calibration points. The results of BI and ML trees all highly supported the monophyly of Heptageniidae and three subfamilies. The phylogenetic relationship of Rhithrogeninae + (Ecdyonurinae + Heptageniinae) was also recovered. The divergence time showed that Heptageniidae originated from 164.38 Mya (95% HPD, 150.23-181.53 Mya) in the mid-Jurassic, and Rhithrogeninae originated from 95.54 Mya (95% HPD, 73.86-120.19 Mya) in the mid-Cretaceous. Ecdyonurinae and Heptageniinae began to diverge at 90.08 Mya (95% HPD, 68.81-113.16 Mya) in the middle Cretaceous. After morphological identification, analysis of the mitogenome's composition, genetic distance calculation, phylogenetic analysis, and divergence time calculation, we suggest that two different populations of Epeorus montanus collected from Aksu, Xinjiang Uygur Autonomous Region (40 degrees 16 ' N, 80 degrees 26 ' E) and Xinyuan, Xinjiang Uygur Autonomous Region (43 degrees 20 ' N, 83 degrees 43 ' E) in China are cryptic species of E. montanus, but further detailed information on their morphological characteristics is needed to fully identify them.
