We examined how Arctic spider (Araneae) biodiversity is distributed at multiple spatial scales in Northern Canada using a standardized hierarchical sampling design. We investigated which drivers, environmental or spatial, influence the patterns observed. Spatial patterns of species richness and composition of Arctic spiders were assessed in 12 sites located in Arctic, Subarctic, and North-Boreal regions, across 30 degrees of latitude and 80 degrees of longitude. Variation of diversity was partitioned in relation to multiple environmental and spatial drivers of diversity patterns. Over 23, 000 adult spiders, representing 306 species in 14 families, were collected in Northern Canada, with 107 species (35% of the total species collected) representing new Territorial or Provincial records. Spider diversity was structured at the regional scale across ecoclimatic regions but not with latitude. Longitudinal patterns of spider diversity across Canada may be explained by post-glacial dispersal. At local scales, diversity was non-randomly distributed, and possibly limited by biotic interactions. We recommend the use of ecoclimatic regions as a framework for conservation of biodiversity in Northern Canada and spiders as useful bioindicators which can help us understand the effects of climate change across ecoclimatic regions of northern Canada.