Behavioral thermoregulation can allow ectotherms to buffer the effects of changes in environmental temperature, and thus an organism's preferred temperature is thought to be under strong selection. However, this contention has seldom been tested. We used common killifish Fundulus heteroclitus from high-latitude (northern) and low-latitude (southern) populations to investigate intraspecific variation in thermal preference and its relationship to habitat temperature. We quantified the preferred temperatures of northern and southern killifish populations acclimated to three temperatures (5 degrees , 15 degrees , and 25 degrees C) to evaluate two alternative hypotheses for the evolution of differences in thermal preference among latitudinally separated populations: local thermal adaptation, which predicts that organisms from high latitudes should prefer lower temperatures than individuals from lower latitudes, versus countergradient variation, which predicts that high-latitude organisms should prefer higher temperatures to compensate for shorter growing seasons. All killifish selected their final thermal preferendum within 4 h. Southern killifish and killifish acclimated to warmer temperatures had greater variability in selected temperature. This increase in variability was the result of an increase in interindividual variation in preferred temperature rather than a reduction in the precision of temperature selection in these groups. Northern killifish preferred significantly higher temperatures than southern fish (30.6 degrees vs. 29.0 degrees C, respectively, when calculated on the basis of the temperature selected consistently for at least 30 min; 28.4 degrees vs. 26.5 degrees C, respectively, when calculated on the basis of the mean temperature occupied), regardless of acclimation temperature. These data are not consistent with local adaptation in thermal preference but instead can be better explained by countergradient variation in thermal preference in killifish.