Quantitative data on the distribution of aquatic vertebrate species along environmental gradients at multiple spatial scales are useful for assessing habitat quality, understanding habitat selection, and predicting the population structures of focal species. In addition, comparable datasets for multiple species enable the examination of the interspecific differences in habitat niches and their differentiation or aggregation patterns in a stream assemblage, as well as the theory of species coexistence. The distributions of aquatic vertebrates and environmental characteristics on longitudinal and microhabitat scales were investigated at 21 sites in a temperate stream, the Yura River, Kyoto, Japan, during the day and at night from August to September 2009. The sites were located from the headwaters to about 40 km downstream along the main channel of the river. Snorkeling observations were conducted using the line-transect method following a standardized procedure at each sampling site with a detailed description of the spatial structure of aquatic vertebrate habitats. The microhabitat environments (water depth, current velocity, substratum characteristics, and presence/absence of vegetation cover) could be determined for all observed individuals. A total of 11,724 individuals belonging to 28 fish, two amphibian, and one reptile species were observed. The data can be used to examine the spatial patterns of focal aquatic vertebrate species and assemblages, and interspecific differences in habitat use among sympatric aquatic vertebrate species at multiple spatial scales.