• Premise of the study: Microsatellite primers were developed for the cardon, a giant columnar cactus (Pachycereus pringlei) of the Sonoran Desert, to investigate intraspecific genetic patterns of diversity and population structure.• Methods and Results: Using 454 GS‐FLX technology and bioinformatics tools, microsatellite primers were successfully identified on 282 reads, including di‐, tri‐, tetra‐, penta‐, and hexanucleotides. A set of 10 primers were characterized on 80 individuals collected in two areas of the Baja California peninsula, Mexico. All 10 loci were polymorphic, with a mean of 6.3 alleles per locus and overall levels of observed and expected heterozygosities ranging from 0.41 to 0.59 and from 0.40 to 0.57, respectively. Alleles per individual plant ranged from one to four, suggesting a polyploidal genome.• Conclusions: These loci should be useful for future investigations of population structure, genetic diversity, and gene flow in the cardon cactus.