Self-incompatibility (SI) is an intraspecific reproductive barrier widely found in flowering plants to prevent self-fertilization but to promote outcrossing. SI is usually controlled by a single polymorphic S-locus. Since the S-loci from several families encode different molecular components regulating SI, they are typically classified into five types (type-1 ~ −5) with type-1 originated from the common ancestor of eudicots and type-2 ~ −5 evolved following the loss of the type-1 S. Both self- and non-self-recognition systems are associated with SI. In self-recognition systems, pollen protein Prp S (Papver rhoeas pollen S) interacts with its cognate stigma Prs S (Papver rhoeas stigmatic S) to initiate a signaling pathway leading to programmed self-pollen death (Papaveraceae, type-3), or pollen protein SCR/SP11 (S-locus cystein-rich/S Pollen 11) interacts with its cognate stigma SRK (S-locus Receptor-like Kinase) leading to destruction of proposed pollen compatibility factors resulting in self-pollen rejection (Brassicaceae, type-2). In non-self-recognition system found in the Solanaceae, Plantaginaceae, Rosaceae, and Rutaceae (type-1), pollen-expressed multiple SLFs (S-locus F-box) form functional SCF complexes to degrade non-self S-RNases allowing cross-pollen growth. Recently, in dimorphic Primula (type-4) and Turnera (type-5), the S-locus encodes several hemizygous genes to control SI, but little is known about their roles in self-pollen rejection. Together, these findings indicate that flowering plants have adopted multiple mechanisms to inhibit self-pollen growth but allow cross-pollen fertilization. In this chapter, we highlight our knowledge about the genetic and molecular regulation of SI responses in flowering plants and several future challenges in addressing their detailed molecular mechanisms and evolution.