Abstract Since self-compatibility has become the primary objective of most almond breeding programmes, search for new self-compatibility sources has acquired a great importance in almond research. The local Spanish cultivar ‘Vivot’, identified as showing the genotype S23Sf, thus presumably self-compatible, was found to be unexpectedly self-incompatible in spite of the presence of the Sf allele, as also observed in other almond cultivars. However, not only the coding sequences of both the Sf-RNase and the SFBf of ‘Vivot’ and ‘Blanquerna’, a confirmed self-compatible cultivar, were identical, but also the 5′ regulatory sequence of the Sf-RNase of both. Thus, the reason for the different expression of the Sf is independent of the complete genetic identity found in the whole chromosome region bordering the S-locus in the almond cultivars sharing the Sf allele.