Cenchrus ciliaris L. is an important perennial forage grass that grows throughout the semi-arid tropics. It reproduces predominantly by apomixis, which provides a means of clonal propagation through seeds. The absence of sexual reproduction in C. ciliaris limits the possibilities of genetic improvement by hybridization. A rare obligate sexual plant of C. ciliaris (IGFRI-CcSx-08/1) that is self-incompatible in nature requires vegetative propagation to maintain its genotype. In vitro culture is one of the possible ways for clonal multiplication and a prerequisite for genetic manipulation. Here, we report high frequency in vitro plant regeneration via callus induction from immature inflorescences of this obligate sexual plant. Embryogenic calli were induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (3.5 mg L-1), and shoot organogenesis was obtained on MS medium supplemented with kinetin (2.0 mg L-1). Rooting from the regenerated shoots was achieved on MS medium containing indolebutyric acid (2.0 mg L-1) and activated charcoal (2.0 g L-1). The survival rate of the plants under ex vitro conditions was 70%. Molecular analysis of the tissue cultured plants using sequence characterized amplified region (SCAR) markers and by embryo sac analysis revealed obligate sexual reproduction in all the regenerated plants. The tissue cultured plants were true to the mother plant type. The high frequency of in vitro plant regeneration achieved by this protocol would be very useful for clonal multiplication and genetic manipulation of this rare genotype of C. ciliaris.