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We used data from 17 to 20 microsatellite markers to investigate the incidence of multiple paternities in wild Agassiz’s desert tortoises, Gopherus agassizii. Neonates were sampled from clutches of eggs laid by wild mothers in nesting enclosures at Edwards Air Force Base and at the Marine Corps Air Ground Combat Center, California. We genotyped 28 clutches from 26 females sampling an average of six neonates per clutch. The number of paternal alleles was used to determine the minimum number of sires for each clutch. Based on conservative criteria requiring evidence from at least two loci to determine multiple paternity, a minimum of 64% of females were polyandrous, while a minimum of 57% of clutches were sired by multiple males. This formed one of the highest incidences of multiple paternities recorded to date in any species of tortoise. The high number of microsatellite loci involved in the analyses allowed detection of multiple paternities in clutches where this may have been missed if fewer loci were used. Our results highlighted the potential pitfalls of quantitatively comparing paternity studies based on differing sampling strategies. Finally, we summarized the conservation implications of the high rate of multiple paternities in this threatened species.