We explored the Theobroma cacao genome sequence to progress in the knowledge of the T. cacao incompatibility system. Cocoa self-compatibility is an important yield factor and has been described as controlled by a late gameto-sporophytic system involving several locus, and resulting in gametic non-fusion. In this work, we identified two different mechanisms controlling the T. cacao self-incompatibility system at two separate loci, located on chromosome one and four (CH1 and CH4). Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to gamete fusion and independently of CH4 locus. Fine mapping and genome wide association studies focused analyses of restricted regions without recombinant plants where several candidate genes were identified. Their expression analysis showed differential expression during incompatible or compatible reactions for some of them. Highly polymorphic SSR diagnostic markers, designed in the CH4 region identified by fine mapping, allowed the development of efficient diagnostic markers predicting selfcompatibility and fruit setting according to allele or genotype presence. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified allowing screening for self-compatible plants in cocoa populations.