Lack of breakthrough in the yield is demanding drought tolerance and adaptation to the harsh arid environment by the released hybrids of pearl millet [Pennisetum glaucum (L) R.Br.]. Fertility restoration patterns of Fl hybrids developed from crosses between male sterile (MS) lines possessing different cytoplasmic MS sources and a genetically diverse set of inbreds were exploited to circumvent this problem. In this paper, we report the extent of genetic diversity and relationship among 20 newly developed MS lines in P. glaucum using nuclear ribosomal DNA and RAPD polymorphism. High level of polymorphism (82.7%) among MS lines of P. glaucum by potential RAPD markers and' with greater proportion of total genetic variation within population (87%) rather than among population (13%) revealed rich genetic diversity among the MS lines developed. Total gene diversity exhibited high levels of variability (H-T=0.2337) and coefficient of gene differentiation (G(st)=0.4651). Phylogram based on multiple sequences further delineated all the 20 MS lines with significant bootstrap values. SNPs at 16 places in ITS-1 are clear indication of phylogenetic utility of ITS-1 region, in particular, in developing bar code of pearl millet germplasm. The MS line CZMS-21 recorded with desirable traits like significantly higher grain yield and the maximum panicle harvest index was identified as a potential source of A4 cytoplasm for the development of stable single cross hybrid for arid environment. The genetic diversity among these MS lines with adaptation to arid environment shall be of significance in developing region specific hybrids.