A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript assembly contigs (TACs) with an N50 of 1510 bp, the largest one being ~8 kb. Of the 21 434 TACs, 16 622 (77.5%) could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions (ISRs). By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping positions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content (PIC) value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.