Knowledge of internal hybridization barriers is relevant for germplasm conservation and utilization. The two pre-zygotic barriers are pollen–pistil self-incompatibility (SI) and cross-incompatibility (CI). To ascertain whether SI and CI were phenotypically related phenomena in potatoes, extensive intra- and interspecific, both intra- and interploidy breeding relationships were established, without previous assumptions on the compatibility behavior of the studied germplasm. Pollen–pistil relationships were analyzed at the individual genotype/accession/family level. In two seasons, 828 intra- and interspecific genotypic combinations were performed, using accessions of the wild potatoes Solanum chacoense Bitter (2n = 2x = 24), S. gourlayi Hawkes (2n = 2x = 24; 2n = 4x = 48), and S. spegazzinii Bitter (2n = 2x = 24), full-sibling (hereinafter “full-sib”) families (2n = 2x = 24) within/between the latter two diploids, and S. tuberosum L. (2n = 4x = 48) cultivars. Pollen–pistil incompatibility occurred in the upper first third of the style (I1/3) in all selfed diploids. In both the intra- and interspecific combinations, the most frequent relationship was compatibility, followed by I1/3, but incompatibility also occurred in the stigma and the style (middle third and bottom third). We observed segregation for these relationships in full-sib families, and unilateral and bilateral incompatibility in reciprocal crosses between functional SI genotypes. Cross-incompatibility in potatoes is, apparently, controlled by genes independent of the S-locus or its S-haplotype recognition region (although molecular evidence is needed to confirm it), with segregation even within accessions.