1. The valley of Cuatro Ciénegas in Coahuila, Mexico has the highest degree of local endemism of any habitat in North America. Despite the arid desert climate, the Cuatro Ciénegas basin harbors an extensive system of permanent streams, wetlands, and spring fed pools.

2. Microsatellite loci were surveyed to infer genetic diversity and population structure of Gambusia marshi, a poeciliid fish endemic to the desert springs of the Cuatro Ciénegas basin and its outflow the Río Salado de Nadadores in Mexico.

3. The Sierra de San Marcos and long-term isolation have played a vital role in shaping the current pattern of phylogeographic structure in G. marshi, and Bayesian clustering analysis revealed four genetic populations within G. marshi as well as a major east-west division of the of the species corresponding to the Sierra de San Marcos.

4. Most sample sites exhibited high levels of genetic differentiation, although there is evidence for recent gene flow between some of these locations, presumably facilitated by irrigation canals. Although all populations exhibit genetic differentiation, the population of G. marshi in Poza Anteojo is extremely divergent and should be considered as an independent management unit.

Gambusia marshi individuals were sampled from seven locations in the Cuatro Ciénegas basin and Río Salado drainage of Coahuila, Mexico. Total genomic DNA was extracted from a 1-2 mm section of fin using the QIAGEN DNeasy blood and tissue extraction kits (QIAGEN, Valencia, CA, USA) following the protocol outlined for DNA isolation from animal tissues. Polymerase chain reaction (PCR) amplification of microsatellite loci was performed using a three-primer method for each locus. The 5’ end of the forward primer of each pair was modified by adding the sequence tag (5’-AGG GTTTTCCCAGTCACGACGTT-3’). This primer was used in conjunction with the unmodified reverse primer and a third primer, identical to the tag on the forward primer, but fluorescently labeled.

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