Powdery mildew (PM) in Cannabis sativa is most frequently caused by the biotrophic fungus Golovinomyces ambrosiae. Based on previously characterized variation in susceptibility to PM, biparental populations were developed by crossing the most resistant cultivar evaluated, 'FL 58', with a susceptible cultivar, 'TJ's CBD'. F1 progeny were evaluated and displayed a range of susceptibility, of which two were self-pollinated to generate two F2 populations. In 2021, the F2 populations (n = 706) were inoculated with G. ambrosiae and surveyed for PM disease severity. In both F2 populations, 25% of the progeny were resistant, while the remaining 75% showed a range of susceptibility. The F2 populations, as well as selected F1 progeny and the parents, were genotyped with a single nucleotide polymorphism array and a consensus genetic map was produced. A major effect quantitative trait locus (QTL) on C. sativa chromosome 1 (Chr01) and other smaller effect QTL on four other chromosomes were identified. The most associated marker on Chr01 was located near CsMLO1, a candidate susceptibility gene. Genomic DNA and cDNA sequencing of CsMLO1 revealed a 6.8 kb insertion in 'FL 58', relative to 'TJ's CBD', of which 846 bp were spliced into the mRNA transcript encoding a premature stop codon. Molecular marker assays were developed using CsMLO1 sequences to distinguish PM resistant and susceptible genotypes. These data support the hypothesis that a mutated MLO susceptibility gene confers resistance to PM in C. sativa and provides new genetic resources to develop resistant cultivars.