Relative growth is often used as a phenotypic measure to distinguish mutant and wild-type yeast or bacterial strains. Differential growth as a function of temperature is a convenient and accurate means of analyzing differences between strains. Slight differences in the genotypes of two strains frequently result in differential growth of the two strains as a function of temperature. We have developed a chamber for the simultaneous growth of multiple strains in microtiter plates along a temperature gradient. Image analysis was used to determine colony area and number at various times as a function of temperature. This chamber reduces the time required and increases the accuracy in measuring growth as a function of temperature. This occurs by allowing relative growth to be measured along a temperature gradient where all other conditions are constant. Two strains of yeast (Saccharomyces cerevisiae) with a known difference in temperature dependence of growth were used to demonstrate the performance of this chamber.