We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.