The most chilling-sensitive stage of rice has been found to be at the onset of microspore release. The microsporocytes produce a wall of callose between the primary cell wall and the plasma membrane, and it has been shown that precise regulation of callose synthesis and degradation in anther is essential for fertile pollen formation. In this study, genes for 10 callose synthases in the rice genome were fully annotated and phylogenetically analyzed. Expression analysis of these genes showed that OsGSL5, an ortholog of microsporogenesis-related AtGSL2, was specifically expressed in anthers, and was notably downregulated by cooling treatment. Gene expression profiles of Rho-type small GTP-binding proteins in rice anther were also analyzed. The mechanisms of callose synthesis in rice pollen formation and its relationships with cool tolerance are discussed.