Most of our understanding of folate metabolism in the parasiteLeishmaniais derived from studies of resistance to the antifolate methotrexate (MTX). A chemical mutagenesis screen ofL.majorFriedlin and selection for resistance to MTX led to twenty mutants with a 2- to 400-fold decrease in MTX susceptibility in comparison to wild-type cells. The genome sequence of the twenty mutants highlighted recurrent mutations (SNPs, gene deletion) in genes known to be involved in folate metabolism but also in novel genes. The most frequent events occurred at the level of the locus coding for the folate transporter FT1 and included gene deletion and gene conversion events, as well as single nucleotide changes. The role of some of theseFT1point mutations in MTX resistance was validated by gene editing. The geneDHFR-TScoding for the dihydrofolate reductase-thymidylate synthase was the second locus with the most mutations and gene editing confirmed a role in resistance for some of these. The pteridine reductase genePTR1was mutated in two mutants. The episomal overexpression of the mutated versions of this gene, but also ofDHFR-TS, led to parasites several fold more resistant to MTX than those overexpressing the wild-type versions. Genes with no known link with folate metabolism and coding for a L-galactolactone oxidase or for a methyltransferase were mutated in specific mutants. Overexpression of the wild-type versions of these genes in the appropriate mutants reverted their resistance. Our Mut-seq approach provided a holistic view and a long list of candidate genes potentially involved in folate and antifolate metabolism inLeishmania.