Physiological constraints can limit thermal niche breadth in organisms, particularly for small-bodied ectotherms. Daily temperature fluctuations often surpass annual (seasonal) temperature variation in the tropics, suggesting diel temperature cycles could drive thermal specialization by individuals that are active at different times of the day. We used foraging workers of the Neotropical ant Ectatomma ruidum to assess whether diurnally and nocturnally active workers differed in thermal tolerance. We compared critical thermal maxima (CTmax) of nocturnal and diurnal foraging workers to explore thermal niche specialization over the diel cycle. We predicted that diurnally active workers would have higher CTmax because they occupy a warmer thermal niche. As predicted, diurnal foragers exhibited significantly higher CTmax (mean 1.1 °C difference) than nocturnal foragers. Diurnal and nocturnal foragers were similar in body size, and there was no relationship between worker size and CTmax. We discuss possible mechanisms for this pattern, and the implications of within- versus between-colony differences in CTmax.