Defining approaches that limit the debilitating consequences of aging is a major challenge of our time. One approach toward improving mid- and later life health focuses on pharmacological interventions that optimize healthy aging across a diverse population. The Caenorhabditis elegans Intervention Testing Program (CITP) formed to study the effects of promising chemicals across diverse genetic backgrounds as a means to identify robust candidates likely to target conserved processes. To test the reproducibility of these lifespan studies, we assessed longevity in 22 Caenorhabditis nematode natural isolates spanning 3 species with multiple biological replicates across 3 laboratories. Although our analyses attributed virtually no variation among the locations, at each site ~10% of observed variation was associated with individual trials, with C. briggsae isolates in particular displaying large variations from trial-to-trial. We next tested 10 chemicals previously reported to affect longevity across a genetically diverse subset of these strains. We found that the reported dietary restriction mimetics in our set of chemicals promoted longevity in the C. elegans strains, but as a group exhibited inconsistent effects across the other Caenorhabditis strains. In contrast, the common laboratory dye ThioflavinT showed generally potent and robust positive effects on lifespan across the Caenorhabditis genus. Our survival analysis results indicated that assessment of genetic and experimental sources of variation is important for the identification of compounds with robust effects on longevity. Our results further highlight specific chemicals that warrant further exploration as potential leads for pharmacological interventions that can improve health in aging animals.