The endemic silverside fish C. humboldtianum is of great ichtyologycal, economical and cultural relevance in central Mexico and it has been suggested that it is among a group of other "peces blancos", the most ancestral species. Here we characterized a set of 26 microsatellite loci from the species in order to further assess population and phylogeographic issues that aid in evaluating their highly impacted populations. From 58 primer pairs tested on specimens from Villa Victoria dam (Rio Balsas Basin), 26 loci positively amplified on the species and cross-primed on specimens of the closely related and threatened Poblana alchichica, C. promelas and C. riojai. Loci resolved medium and high genetic variation (mean NA = 14.3, SD = 9.38; mean HO & HE = 0.47, SD = 0.32 and * Corresponding author. B.-S. I. de los Angeles et al. 356 0.58, SD = 0.32, respectively) and it is expected that these polymorphic loci are also useful in studing threatened atherinopsid species.