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Apospory in Pennisetum squamulatum Fresen is inherited as a single-locus dominant trait. Molecular analysis has identified the apospory-specific genomic region (ASGR), a large segment of one chromosome that is necessary and sufficient to confer apomixis. To transfer apospory from P. squamulatum to pearl millet [P. glaucum (L.) R. Br.], advanced backcrosses were generated. Here we report the characterization of apomictic [BC.sub.7] and [BC.sub.8] generations. A [BC.sub.7]-derived genotype has two P. squamulatum chromosomes, while [BC.sub.8]-derived apomictic lines inherited only the ASGR chromosome that confers apospory to pearl millet. Morphologically, no significant differences were observed between backcross generations for plant height, leaf length, leaf width, and first internode and inflorescence lengths. However, the [BC.sub.7] and [BC.sub.8] lines differed significantly with regard to pollen viability, flowering time, and seed set. Pollen viability increased to 81% in the [BC.sub.8] lines compared with 37% in [BC.sub.7]. The frequency of aposporous embryo sacs was lower in [BC.sub.8] (79%) lines compared to [BC.sub.7] (93%). Meiotic behavior of chromosomes during metaphase I and anaphase I showed that [BC.sub.8] typically had a single univalent, identified as the ASGR chromosome by FISH (fluorescence in situ hybridization) and GISH (genomic in situ hybridization). Utilizing its unique behavior during meiosis the ASGR chromosome was microdissected and a chromosome-specific library was constructed and sequenced. Analysis of sequences identified both novel and previously known ASGR-linked sequences. doi: 10.2135/cropsci2009.05.0263