We examined the diversity of Lepidoptera in several Ohio natural areas at local and regional scales. First, we conducted a field study to assess effects of spatial and temporal extent on species accumulation in inventories of forest moth communities in the North Central Tillplain Ecoregion. We then compared the complementarity among moth communities found in nature reserves in two other Ohio ecoregions to the moth community described from the North Central Tillplain. As expected, more moth species were inventoried when multiple sites and multiple years were examined. Species accumulation curves did not saturate for any inventory, regardless of spatial or temporal extent. Within the North Central Tillplain, nature reserves of similar size and floristic composition supported equally rich moth communities but species turnover among reserves suggested that floristically similar habitats supported different species assemblages. Species turnover estimates were not significantly different between macro- and microlepidoptera; rarity was more important in determining community heterogeneity than coarse taxonomic affiliation. At a regional scale, nature reserves in different Ohio ecoregions had a species complementarity of 50–70%, indicating significant turnover of species across broad scales. This suggests that considerable heterogeneity exists among moth communities at both local and regional scales. Successful conservation of Lepidoptera in fragmented landscapes should strive to represent the natural heterogeneity of species richness at both local and regional scales.