Varroa destructor has been present in Cuba since 1996, but without the use of acaricidal infestation rates remain at very low levels. The presence of Korean haplotype mites was described in 2007, but there is no information regarding the introgression of the less virulent Japanese haplotype that could account for a low pathogenicity of the mite. In this research, we carried out molecular characterization of Cuban Varroa mites through mitochondrial DNA and hypervariable nuclear loci. We applied an alternative RFLP technique and found that all the analyzed samples corresponded to Korean haplotypes. We analyzed the three STRs loci VD112, VD114 and VD016, previously described as highly variable and found new alleles in all of them, with an absolute allele size very different to those reported worldwide. We also detected genic and genotypic differentiation between samples from two nearby locations (P=0.08). We also tested a new RFLP method for mite haplotype discrimination with an intra-reaction positive control of digestion.