A diploid potato recombinant inbred line population was derived from a cross between Solanum chacoense inbred line M6 and S. chacoense accession USDA8380-1 (80-1) to identify loci associated with self-compatibility and Colorado potato beetle resistance. Individuals from the F4 and F5 generations were genotyped on the Illumina Infinium V3 22K Single Nucleotide Polymorphism (SNP) Array and a genetic map constructed. All F5 individuals contain at least one copy of the dominant S-locus inhibitor (Sli) haplotype; however, not all F5 individuals set fruit. Pollen tubes reached the ovaries of both self-fruitful and self-unfruitful plants, indicating that the presence of the dominant Sli allele is not sufficient for selfed fruit and seed production. Loci on chromosomes 3, 5, 6 and 12 were identified as novel targets for self-fertility improvement. Evaluation of fruit and seed set upon selfing in the F4 generation over two greenhouse seasons revealed environmental influence on self-fertility. Loci exhibiting residual heterozygosity were found on all chromosomes except chromosomes 3 and 11 in F5 inbreds, but none of the measured self-fertility traits were correlated with the level of heterozygosity. Four SNPs on chromosome 2 between 22,151,711-22,381,719 bp were associated with foliar leptine glycoalkaloid synthesis and Colorado potato beetle resistance in the recombinant inbred line population. Robust inbred lines carrying Colorado potato beetle resistance were developed without field selection during the inbreeding process and beetle resistance was introgressed into diploid breeding lines.