Raffinose synthase(Rafs) is an important enzyme in the synthesis pathway of raffinose from sucrose and galactinol in higher plants and is involved in the regulation of seed development and plant responses to abiotic stresses. In this study, we analyzed theRafsfamilies and profiled their alternative splicing patterns at the genome-wide scale from 10 grass species representing crops and grasses. A total of 73Rafsgenes were identified from grass species such as rice, maize, foxtail millet, and switchgrass. TheseRafsgenes were assigned to six groups based the phylogenetic analysis. We compared the gene structures, protein domains, and expression patterns ofRafsgenes, and also unraveled the alternative transcripts of them. In addition, different conserved sequences were observed at these putative splice sites among grass species. The subcellular localization ofPvRafs5suggested that theRafsgene was expressed in the cytoplasm or cell membrane. Our findings provide comprehensive knowledge of theRafsfamilies in terms of genes and proteins, which will facilitate further functional characterization in grass species in response to abiotic stress.