We have estimated the extent of genetic variation in museum (1890s) and contemporary (1980s) samples of Florida panthers Puma concolor coryi for both nuclear loci and mtDNA. The microsatellite heterozygosity in the contemporary sample was only 0.325 that in the museum samples although our sample size and number of loci are limited. Support for this estimate is provided by a sample of 84 microsatellite loci in contemporary Florida panthers and Idaho pumas Puma concolor hippolestes in which the contemporary Florida panther sample had only 0.442 the heterozygosity of Idaho pumas. The estimated diversities in mtDNA in the museum and contemporary samples were 0.600 and 0.000, respectively. Using a population genetics approach, we have estimated that to reduce either the microsatellite heterozygosity or the mtDNA diversity this much (in a period of c. 80years during the 20th century when the numbers were thought to be low) that a very small bottleneck size of c. 2 for several generations and a small effective population size in other generations is necessary. Using demographic data from Yellowstone pumas, we estimated the ratio of effective to census population size to be 0.315. Using this ratio, the census population size in the Florida panthers necessary to explain the loss of microsatellite variation was c .41 for the non-bottleneck generations and 6.2 for the two bottleneck generations. These low bottleneck population sizes and the concomitant reduced effectiveness of selection are probably responsible for the high frequency of several detrimental traits in Florida panthers, namely undescended testicles and poor sperm quality. The recent intensive monitoring both before and after the introduction of Texas pumas in 1995 will make the recovery and genetic restoration of Florida panthers a classic study of an endangered species. Our estimates of the bottleneck size responsible for the loss of genetic variation in the Florida panther completes an unknown aspect of this account. ?? 2008 The Authors. Journal compilation ?? 2008 The Zoological Society of London.