Simple SummaryPatterns of variation within a widespread species can provide evidence of population history. Adjacent stable populations with gene flow show clinal genetic divergence forming a pattern of isolation by distance. Populations that have grown due to an increase in potential habitat such as forest cover since the last glacial maximum will have low genetic variation showing patterns of range expansion. The mayfly Acanthophlebia cruentata of Aotearoa, New Zealand, is widespread in streams of North Island but absent from the cooler South Island. Mayfly nymphs are restricted to streams but adults fly, facilitating gene flow among catchments. We detected higher genetic diversity at lower latitudes of this mayfly’s range compared to most of its distribution, concordant with predictions of limited forest cover in New Zealand during Pleistocene glacial periods. A signature of recent range expansion was observed in the higher latitudes. Despite initial observation suggesting mayfly size correlated with latitude, we found sex, elevation and sampling date were significant predictors of size, and some size variation is also explained by three regional groups based on haplotype distribution.The mayfly Acanthophlebia cruentata of Aotearoa, New Zealand, is widespread in Te Ika-a-Māui North Island streams, but has never been collected from South Island despite land connection during the last glacial maximum. Population structure of this mayfly might reflect re-colonisation after volcanic eruptions in North Island c1800 years ago, climate cycling or conceal older, cryptic diversity. We collected population samples from 33 locations to estimate levels of population genetic diversity and to document phenotypic variation. Relatively low intraspecific haplotype divergence was recorded among mitochondrial cytb sequences from 492 individuals, but these resolved three geographic-haplotype regions (north, west, east). We detected a signature of isolation by distance at low latitudes (north) but evidence of recent population growth in the west and east. We did not detect an effect of volcanic eruptions but infer range expansion into higher latitudes from a common ancestor during the last glacial period. As judged from wing length, both sexes of adult mayflies were larger at higher elevation and we found that haplotype region was also a significant predictor of Acanthophlebia cruentata size. This suggests that our mitochondrial marker is concordant with nuclear genetic differences that might be explained by founder effect during range expansion.