Although all species of the Brachypelma genus are protected under CITES, few studies have been performed on the genetic structure of the populations of these endangered tarantulas. Here we propose, for the first time in spiders, to use ISSR (Inter Simple Sequence Repeat) technique to study the genetic variability of Mexican populations of Brachypelma vagans (Ausserer 1875). We used a nonlethal technique to collect samples from six populations in the Yucatan peninsula and we tested seven ISSR primers. Four of these primers gave fragments (bands) that were sufficiently clear and reproducible to construct a binary matrix and determine genetic variability parameters. We revealed a very high percentage of polymorphism (P  =  98.7%) the highest yet reported for tarantula spiders. Our results show that the ISSR-PCR method is promising for intraspecific variation of tarantula spiders.