The American horseshoe crab, Limulus polyphemus, is found along the Atlantic and Gulf of Mexico coasts in genetically-isolated populations. Eggs are laid in shoreline beaches that expose developing embryos to combinations of environmental stressors. Whether populations of L. polyphemus differ in multi-stressor tolerance had never been tested. We assessed the multi-stressor tolerance of L. polyphemus embryos from a population in Delaware Bay (DE) and determined whether these differed from the multi-stressor tolerance of embryos from a more southerly Florida Gulf Coast (FGC) population. We monitored the field sediment temperatures and determined multi-stressor tolerance of DE embryos, then compared these to published data for FGC embryos. For multi-stressor tolerance, we assessed development success of embryos in two-week exposures to 36 fully-factorial combinations of temperature (20, 25, 30, 35 °C), salinity (5, 15 and 34 ppt) and ambient O2 (5, 13 and 21% O2), followed by two-weeks in recovery conditions. Sediment temperatures in the DE site ranged from 9.5-46 °C, with extended periods exceeding 35 °C. Development success was similar between the DE and FGC populations in 14 of 26 multi-stressor combinations. The DE embryos were generally more successful in conditions that included high temperature or moderate hyposalinity, whereas the FGC embryos were generally more successful in conditions that included extreme hyposalinity. This suggests that although multi-stressor tolerances are generally similar between the two populations, specific differences exist that correlate more with differences in nest microenvironment than latitude.