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The efficacy of sweet cherry production is highly dependent on the regularity of flowering events and genetic-determined relations between female sporophyte and male gametophyte, which became even more important with higher flowering temperatures caused by climate change. Special attention is paid to the genetic diversity that provides essential sources of potential temperature-tolerance genes. Our study aimed at the genetic and reproductive characterization of Balkan cherry cultivars of autochthonous origin (‘Canetova’, ‘G-2’, ‘Dolga Šiška’ and ‘Ohridska Crna’), and six potential pollenizers. To identify S-haplotypes, the polymerase chain reaction (PCR) method was used to detect the S-ribonuclease (S-RNase) and S-haplotype-specific F-box protein (SFB) alleles, combined with fragment analysis and S-RNase sequencing. Pollination experiments were performed at three Balkan localities over two flowering seasons, and the fluorescence microscopy method was used to assess the cultivars’ male/female reproductive behaviour. A novel S-RNase allele S₄₀ was identified in ‘Ohridska Crna’ for the first time. ‘Ohridska Crna’ also demonstrated the best adaptability to higher temperatures regarding primary ovule longevity. This feature makes it desirable from the aspect of breeding new cultivars that can withstand the impacts of climate change. The findings on male-female relations and their temperature dependence open up the possibility for yield prediction and smart horticultural decisions that can be made to guide cherry production. Full article