Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.