Plant breeders continue their search for an effective and efficient method for the identification of drought tolerance in large segregating populations. In the past, physiological based assays have been too time consuming to be applied to breeding programs. Plant physiologists have found chlorophyll fluorescence (CF) to be a valuable tool to monitor plant stress response. The objectives of this field study were to [1] determine the repeatability of CF measurement in field grown upland cotton [2] determine if an array of genotypes could be classified according to their CF readings, and [3] evaluate the feasibility of CF as a tool for drought tolerance evaluation in cotton breeding programs. A drought bioassay has been developed utilizing CF to monitor plant cell viability under dark, high temperature conditions. Leaf punches are harvested at predawn since water-stressed plants will not mobilize carbohydrate reserves overnight so source leave samples will maintain higher levels of CF under high temperature dark incubation, with the opposite being true for non-stressed plants. This assay was used to evaluate 20 genotypes at two locations over two years. The experiment was a split plot design of a randomized complete block with irrigation treatment as main plots and genotypes as subplots. The data indicate that variability exists for CF among the 20 genotypes tested. Rankings are especially consistent at the high and low ends of the mean separation for CF values. Future tests of correlation between CF values and lint yield may further support the use of CF evaluation for drought tolerance in cotton breeding programs.