Cultivated diploid potatoes (2n = 2x = 24) are self-incompatible, but can be altered to become self-compatible using the Sli gene. Previously, a diploid clone 97H32-6 was selfed up to S3 using the Sli gene. To explore the usefulness of the Sli gene for the production of highly homozygous diploid potatoes, 2 S4 families from the above 97H32-6 derived S3 lines (inbred series A) and 3 S5 families by continuous selfings from a different F1 (= S0) plant (inbred series B) were developed. The level of heterozygosity and the location of heterozygous loci on the genetic map were investigated using RFLP and AFLP markers. The average heterozygosity levels of the originally heterozygous loci decreased from 100% in S0 to 10.7% in S4 and 8.6% in S5 (inbred series A and B, respectively). The average rate of reduction in heterozygosity per generation (38.4% and 38.5% for inbred series A and B, respectively) was lower than the theoretically expected rate (50%). However, none of the loci or chromosome sections was exclusively heterozygous in the advanced self-progeny. Thus, highly homozygous and seed-propagated diploid potatoes could be obtained by repeated selfing using the Sli gene.