Eastern sand darter (Ammocrypta pellucida) is listed as Threatened under the Canadian Species at Risk Act. Little relevant biological information is available for most Canadian populations and only limited information is available for populations in the United States. To supplement the paucity of information, this study collected biological information on eastern sand darter during field surveys in 2006–2007 from 10 sites in the lower Thames River, Ontario, Canada. Collected data were used to estimate critical life history traits including: longevity, fecundity, clutch size and number, growth, survival, age-at-first-maturity and cohort age structure. Longevity was 3+ years, with age-at-first-maturity being 1+ for both sexes. Examination of egg sizes from preserved females in 2007 showed a bimodal distribution, suggesting two spawning occurrences of 71 eggs each. Quantitative comparison of lower Thames River biological information with a more southerly eastern sand darter population in the Little Muskingum River, Ohio, demonstrated little variation between the populations. Data comparisons suggest that localised environmental factors are affecting biological traits (growth), in particular water temperature that may be controlled by differences in riparian cover and/or groundwater input.