Cross-compatibility behaviour of doubled haploid (DH) and hybrid genotypes of Coffea camphora was established using both phenotypic bioassay and in situ seed-set examination. The availability of DHs provided the opportunity of working with genetically homogenous pollen and female parents. The aniline blue fluorescence (ABF) method was applied to detect callose accumulation in pollen and pistil. Clear cross-compatibility/incompatibility situations were observed and confirmed by in situ seed-set analysis. Cross-compatibility analysis of hybrid combinations involving different DHs corroborated the crossing behaviour observed at the DH level. Expression of the self-incompatibility system did not appear to be affected by the low vigour of the DH. The crossing-behaviour distribution observed within DHs derived from clone IF200 confirmed that self-incompatibility in C. canephora is a gametophytic self-incompatibility system controlled by a single locus (S-locus). Reduced seed-set developments following incompatible crosses may indicate the occurrence of pseudo-incompatibility. Molecular marker linkage analysis showed that the S-locus is associated with an RFLP marker on linkage group 9. The availability of a linked DNA marker should facilitate the genetic analysis of self-incompatibility in relation to coffee breeding programmes.