Simple Summary Understanding the distribution of plants and animals is a major focus of ecologists and is of increasing importance as temperatures rise and the world's climate becomes more unpredictable. One large-scale pattern that has often been observed is for the number of species in a group of interest to be greater in the tropics than towards the poles. Species also tend to decline in number at higher elevations. We examined the distribution of mayfly species, insects commonly found in streams, at multiple sites throughout New Zealand to see whether the numbers and identities of species were related to latitude and altitude. We found that the number of mayfly species declined from north to south, i.e., towards higher latitudes, and less strongly as elevation increased. Furthermore, the species composition of communities became increasingly different with distance along the latitudinal gradient. Our findings suggest that the species gradient has historical origins, with the north of North Island and the South Island mountains likely to have been important regions of speciation 1.5-0.5 million years ago.Abstract The distribution of species in relation to latitude and altitude is of fundamental interest to ecologists and is expected to attain increasing importance as the Earth's climate continues to change. Species diversity is commonly greater at lower than higher latitudes on a global scale, and the similarity of communities frequently decreases with distance. Nevertheless, reasons for such patterns are not well understood. We investigated species richness and changes in community composition of mayflies (Ephemeroptera) over 13 degrees of latitude at 81 locations throughout New Zealand by light-trapping and the benthic sampling of streams. Mayflies were also sampled along an altitudinal gradient on a prominent inactive volcano in the east of North Island. Sampled streams were predominantly in the native forest, at a wide range of altitudes from sea level to c. 1000 m a. s. l. A total of 47 of the 59 described New Zealand mayflies were recorded during the study, along with five undescribed morphospecies. Species richness declined and the degree of dissimilarity (beta diversity) of mayfly communities increased significantly from north to south but less strongly with increasing altitude. Our results suggest that the southward decline in species richness has historical origins with the north of the country having acted as a major refuge and region of speciation during the Pleistocene. The increasing dissimilarity of the northern and southern communities may reflect an increasingly harsh climate, variable amounts of subsequent southward dispersal of northern species and, in the South Island, the presence of species which may have evolved in the newly uplifted mountains during the Miocene-Pliocene.