Cocaine use disorders (CUD) are devastating for affected individuals and impose a significant societal burden, but there are currently no FDA-approved therapies. The development of novel and effective treatments has been hindered by substantial gaps in our knowledge about the etiology of these disorders. The risk for developing a CUD is influenced by genetics, the environment and complex interactions between the two. Identifying specific genes and environmental risk factors that increase CUD risk would provide an avenue for the development of novel treatments. Rodent models of addiction-relevant behaviors have been a valuable tool for studying the genetics of behavioral responses to drugs of abuse. Traditional genetic mapping using genetically and phenotypically divergent inbred mice has been successful in identifying numerous chromosomal regions that influence addiction-relevant behaviors, but these strategies rarely result in identification of the causal gene or genetic variant. To overcome this challenge, reduced complexity crosses (RCC) between closely related inbred mouse strains have been proposed as a method for rapidly identifying and validating functional variants. The RCC approach is dependent on identifying phenotypic differences between substrains. To date, however, the study of addiction-relevant behaviors has been limited to very few sets of substrains, mostly comprising the C57BL/6 lineage. The present study expands upon the current literature to assess cocaine-induced locomotor activation in 20 inbred mouse substrains representing six inbred strain lineages (A/J, BALB/c, FVB/N, C3H/He, DBA/2 and NOD) that were either bred in-house or supplied directly by a commercial vendor. To our knowledge, we are the first to identify significant differences in cocaine-induced locomotor response in several of these inbred substrains. The identification of substrain differences allows for the initiation of RCC populations to more rapidly identify specific genetic variants associated with acute cocaine response. The observation of behavioral profiles that differ between mice generated in-house and those that are vendor-supplied also presents an opportunity to investigate the influence of environmental factors on cocaine-induced locomotor activity.