Genetic variability in humans is a key characteristic underlying susceptibility potential for responses to environmental chemicals, yet animal testing paradigms typically do not capture the breadth of human diversity. Diversity Outbred (J:DO) mice are a population of outbred animals with well-randomized genetic sequence polymorphisms and a genetic architecture similar to that of human populations. This study was designed to evaluate baseline characteristics of J:DO mice that could serve as a foundation for future toxicological studies using this population-based model. Adult male DO mice (n = 75) were fed a control diet and monitored for 13 weeks. Clinical chemistry values and calculated reference ranges, organ and body weights, and background histopathology for the liver and kidney are provided. Measurements of sperm parameters and levels of leptin and insulin are included to assess variability in reproductive and endocrine parameters. Power calculations are provided for endpoints having comparator data from prior recent NTP studies, allowing researchers to determine the number of DO mice needed—per endpoint—to observe a comparable statistical difference to that using conventional rodent stocks (80% power, p = 0.05). The data suggest sample sizes for guideline toxicological studies when using DO mice would need to be increased to at least 16–34 mice for most conventional endpoints. In contrast, hormonal and reproductive endpoints would require >100 mice per endpoint to achieve the same power for determining a difference in experimental groups comparable to that of conventional studies, which may impact study design. The results of this initial characterization of DO mice suggest an inherent variability in the underlying biology that might provide utility for evaluating population dynamics in responses relevant to genetically diverse human populations.