Pyxis arachnoides is a Critically Endangered Malagasy tortoise for which little data about its natural history and genetic variation are available. This lack of knowledge complicates undertaking conservation measures considering Pyxis genetic diversity. Here, we describe six new microsatellites for Pyxis arachnoides from wild specimens and several localities, and the cross amplification of two Astrochelys radiata microsatellites. Of these microsatellites, two are monomorphic among the three distinct subspecies and a closely related species, while the remaining six are polymorphic. Microsatellite data are needed for this species to study the inter- and intra-subspecies genetic diversity, especially for the highly endangered subspecies P. a. brygooi, which currently occurs only in remote localities. The availability of polymorphic microsatellites will allow identifying the population of origin of individuals and will aid in justifying their management as separate evolutionary lineages for conservation purposes. The rationale is to preserve both evolutionary processes and adaptive potential.