Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination of this neglected tropical disease. Here we conducted whole genome resequencing, proteome profiling, and comparative analyses of a drug-resistant clinical isolate and two drug-susceptible strains of Leishmania donovani to explore genetic features that might contribute to the establishment of drug resistance in this parasite. By comparative genomic analysis, exclusive variations were identified in the drug-resistant isolate of L. donovani, including 86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes. Comparative proteomic analysis indicated significant differences in protein expression between resistant and susceptible strains of L. donovani, including 69 exclusive detected molecules and 84 consistent down-/up-regulations in the former. Integrating the genomic mutations and proteomic specificities linked nine of the genomic mutations (gene duplication, insertion and deletion) to significantly altered protein expression changes in the drug-resistant clinical isolate. These genetic features were inferred to be associated with nucleotide-binding and fatty acid metabolism (biosynthesis and degradation), which might contribute to fitness-gains allowing for the drug-resistant phenotype of L. donovani. This comparative and integrative work provided deep insights into the molecular basis underlying resistance establishment, suggesting new aspects to be investigated for novel intervention strategies against L. donovani and related species.