We attempted to generate homozygous dhfr-ts (dihydrofolate reductase-thymidylate synthase) knockouts in virulent Leishmania major, an asexual diploid protozoan parasite. Transfection of a neo (neomycin phosphotransferase) targeting fragment yielded heterozygous replacement lines with high efficiency. However, second transfections with a hyg (hygromycin B phosphotransferase) targeting fragment in the presence of metabolites shown to rescue homozygous knockouts in attenuated Leishmania did not yield the expected dhfr-ts- thymidine auxotrophs obtained previously with attenuated lines. Molecular karyotype, Southern blot, and flow cytometric DNA content analysis of clonal transfectants revealed three classes: (i) genomic tetraploids, containing two wild-type dhfr-ts chromosomes and one neo and one hyg replacement chromosome; (ii) aneuploid trisomic lines with one wild-type dhfr-ts and one neo and one hyg replacement chromosome; (iii) diploids bearing homologous integration of the targeting fragment without replacement. Aneuploid and tetraploid lines predominated. This confirms the common impression that natural populations of Leishmania are often aneuploid. The remarkable ability of these parasites to undergo and tolerate changes in chromosome number suggests a general method for testing whether genes are essential for growth in vitro, as the ability of Leishmania to simultaneously undergo homologous gene replacement while retaining wild-type genes by increasing chromosome number provides a diagnostic and positive experimental result. Our results show that virulent Leishmania require at least one copy of dhfr-ts and argue that DHFR-TS plays an unanticipated role in addition to its role in the de novo synthesis of thymidine. These results also have implications for genetic tests of the organization of Leishmania populations.