Resolution of QTL detection with conventional mapping populations is limited as overshadowing by major QTLs prevents estimation of independently segregating minor QTL numbers and interactions between QTLs. Chromosome segment substitution lines (CSSLs) permit precise QTL mapping and evaluation of gene actions and interactions. Identifying molecular markers associated with combining ability for yield traits under moisture stress conditions would facilitate the genetic basis of drought tolerance. The present study estimated the general combining ability (GCA) and specific combining ability (SCA) effects of 15 grain and stover yield-related traits under three different moisture regimes using a set of testcrosses with CSSLs of pearl millet linkage group 1(LG1), and also identified molecular markers associated with combining ability of these traits. GCA and SCA of the 15 grain and stover yield-related traits of the CSSLs were estimated by the performance of testcrosses with three genetically diversified testers. There were significant differences in the GCA and SCA of traits among CSSLs. GCA and SCA of grain yield were correlated with that of panicle harvest index and grain number, while stover yield with biomass and vegetative growth index. A total of four significant loci for GCA and one locus for SCA were identified in at least two moisture regimes at p<0.01, demonstrating that the genetic basis of GCA and SCA is different. The loci for the GCA and SCA identified in multiple moisture regimes in this study would be useful for pearl millet breeding for drought tolerance.