The Chilean abalone aquaculture industry is only supported with two species introduced during the late 1970s: red abalone Haliotis rufescens and Japanese abalone H. discus hannai. At the moment, red abalone accounts for 97% of total production due to its adaptability for full-cycle culture and faster growing than the Japanese species. However, Japanese abalone has a better acceptance and higher prices in Asian markets. These dualities have been merged by the successful hybridization between red and Japanese abalone. The goal of this study was to carry out a genetic analysis on red and Japanese abalone populations and their interspecific hybrids. Microsatellite markers were applied in three hatchery populations (HRed, HJap, and HHyb) to assess the genetic diversity and to certificate hybrid status. Allelic diversity was similar between HRed and HHyb populations (12 alleles), whereas for HJap it was significantly lower (7.8 alleles). Mean observed and expected heterozygosity (H_o, H_e) were 0.533 (0.045) and 0.786 (0.031), with no significant differences among populations (P ≤ 0.05). In most cases, Hovalues were lower than H_e, indicating significant deviations from Hardy-Weinberg equilibrium. Comparison among populations showed that the hybrids are comparatively more similar to H. rufescens than H. discus hannai. Furthermore, hybrid status was confirmed by the presence of species-specific bands for each parental species of microsatellite locus Hco97. This work is the first approach to characterize genetically hybrids of H. rufescens × H. discus hannai produced in Chile.