Using DNA barcode analysis, nest collections, and pan-trapping we compared molecular differences, nesting behaviour, and phenology of three of the four species of Ceratina Latreille present in the Niagara Region of southern Ontario, Canada: C. dupla Say, C. calcarata Robertson, and C. mikmaqi Rehan and Sheffield. Ceratina dupla and C. mikmaqi were separated by five fixed nucleotide differences and an average sequence divergence of 1.86%. In our population, C. mikmaqi and C. calcarata were common and C. dupla was rare. Ceratina dupla nested earlier than C. mikmaqi and C. calcarata, and sometimes produced a second brood in late July – early August. Each species constructed linear nests in the pith of dead twigs, C. mikmaqi and C. dupla usually in Fuller's teasel (Dipsacus fullonum L.; Dipsacaceae) and C. calcarata usually in raspberry (Rubus L.; Rosaceae). Genetically distinct, each species occupies a slightly different niche in the Niagara bee assemblage.