To evaluate the current genetic status of Chilean abalone hatchery populations, we tested 25 heterologous microsatellite loci in Haliotis rufescens Swainson 1822. Successful cross-amplification was obtained with 13 microsatellite loci, and 7 were used to estimate genetic variability in 4 separate hatchery populations: 2 located in Caldera, 1 in Coquimbo (northern Chile), and one more on Chiloe Island (southern Chile). The number of alleles per locus (N_A) ranged from 2–16, with an average of 8.86. Among populations, mean H_o and H_e values were 0.510 and 0.746, respectively, with significant deviations from Hardy-Weinberg equilibrium at 23–28 of the population loci analyzed (82.1%). The F_IS values calculated showed no significant differences among the different hatcheries analyzed. However, F_IS values from the Caldera populations were close to panmixia values, whereas Coquimbo and Chiloe populations showed a slight tendency to endogamy. According to the F_ST and R_ST values, there are genetic differences among the populations, with the 3 northern hatcheries from Caldera and Coquimbo more related than the abalone population of southern Chile. In addition, comparison between California wild and Chilean hatchery abalone populations showed a loss of genetic diversity evidenced by microsatellite markers. The current study contributes to the genetic knowledge of the Chilean red abalone aquaculture resource and reveals the necessity to perform genetic monitoring in hatchery populations.