Resistance to antimonials has emerged as a major hurdle to the treatment and control of visceral leishmaniasis (VL), also know as kala-azar (KA), the disease caused by Leishmania donovani, in India where >60% of KA patients are unresponsive to sodium antimony gluconate (SAG) treatment. Determinants of resistance in laboratory strains are partly known, however the mechanism operating in field isolates is not well understood. In microarray-based expression profiling with RNA isolated from field isolates of drug-resistant and -sensitive L. donovani parasites, genes encoding histone 1 (H1), histone 2A (H2A), histone 4 (H4), mitogen-activated protein kinase 1 (MAPK1) and two hypothetical proteins showed significantly higher expression in antimony-resistant parasites, whilst genes encoding an amino acid transporter showed higher expression in sensitive parasites. The expression level of these genes was validated by semiquantitative polymerase chain reaction (PCR). Furthermore, the higher expression of H1, H2A and MAPK1 was confirmed at the protein level in resistant isolates. Overexpression of H2A in a drug-sensitive laboratory strain as well as a field isolate of L. donovani resulted in conversion of SAG-sensitive Leishmania parasites into a resistant phenotype. Moreover, H2A overexpression resulted in a significant decrease in susceptibility towards other antileishmanial drugs currently in use, i.e. amphotericin B and miltefosine, pointing to its role in drug resistance.