Pennisetum squamulatum reproduces by apomixis, a type of asexual reproduction through seeds. Apomixis in P. squamulatum is transmitted as a dominant Mendelian trait, and a genomic region, the apospory-specific genomic region (ASGR), is sufficient for inheritance of the trait. The ASGR is physically large (>50 Mb), highly heterochromatic, hemizygous, and recombinationally suppressed. These characteristics have hindered high-resolution genetic mapping and map-based cloning of apomixis genes. In this study, the long terminal repeat (LTR) regions of ASGR-abundant retrotransposons in the genome of P. squamulatum and ASGR-linked bacterial artificial chromosome clones were identified and sequenced for designing LTR-specific primers. Two hundred and ninety single-dose sequence specific amplified polymorphism (SSAP) markers were generated from 38 primer combinations. The SSAP markers combined with two previous ASGR-mapped markers were used for genetic linkage analysis and construction of a genetic map resulting in the formation of 27 linkage groups at LOD 10, one of which contained >60% of the SSAP markers. After removing identical markers (identical band scoring) on the largest linkage group, 46 markers were finally used for genetic mapping at LOD 10. The markers distributed across 10 different loci covering 19 cM; however, 45 markers were distributed within 9 cM. Six markers were recovered and sequenced. Five markers were successfully converted into sequence characterized amplified regions (SCARs). Segregation of SCAR markers was not always consistent with the SSAP markers of origin suggesting a greater level of error in the SSAP map resulting in an inflated map distance for the ASGR. One SCAR marker (Pst 56-1205-400) detected expression of an ASGR retrotransposon in root, anther, leaf and ovary of P. squamulatum, although sequencing of the RT-PCR product failed to find a functional open reading frame for the transcript.