Stomatal density and size affect plant water use efficiency, photosynthsis rate and yield. The objective of this study was to gain insights into the variation and genetic basis of stomatal density and size during grain filling under drought stress(DS) and well-watered(WW) conditions. The doubled haploid population derived from a cross of wheat cultivars Hanxuan 10(H10), a female parent, and Lumai 14(L14), a male parent, was used for phenotyping at the heading, flowering, and mid- and late grain filling stages along with established amplified fragment length polymorphism(AFLP) and simple sequence repeat(SSR) markers. The stomatal density of doubled haploid(DH) lines was gradually increased, while the stomatal lengths and widths were gradually decreased during grain filling stage. Twenty additive QTLs and 19 pairs of epistatic QTLs for the 3 traits were identified under DS. The other 20 QTLs and 25 pairs epistatic QTLs were obtained under WW. Most QTLs made more than 10% contributions to the total phenotypic variations at one growth stage under DS or WW. Furthermore, QTLs for stomatal density near Xwmc74 and Xgwm291 located on chromosome 5A were tightly linked to previously reported QTLs regulating total number of spikelets per spike, number of sterile spikelets per spike and proportion of fertile spikelets per spike. Qsw-2D-1 was detected across stages, and was in the same marker region as a major QTL for plant height, QPH.cgb-2D.1. These indicate that these QTLs on chromosomes 5A and 2D are involved in regulating these agronomic traits and are valuable for molecular breeding. Stomatal density and size affect plant water use efficiency, photosynthsis rate and yield. The objective of this study was to gain insights into the variation and genetic basis of stomatal density and size during grain filling under drought stress (DS) and well-watered (WW The double haploid population derived from a cross of wheat cultivars Hanxuan 10 (H10), a female parent, and Lumai 14 (L14), a male parent, was used for phenotyping at the heading, flowering, and mid- and late grain filling stages along with established amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The stomatal density of doubled haploid (DH) lines was gradually increased, while the stomatal lengths and widths were gradually decreased during grain filling stage. Twenty additive QTLs and 19 pairs of epistatic QTLs for the 3 traits were identified under DS. The other 20 QTLs and 25 pairs of epistatic QTLs were obtained under WW. Most QTLs made more than 10% contributions to the total phenotypic variations at one growth stage under DS or WW. QTLs for stomatal density near Xwmc74 and Xgwm291 located on chromosome 5A were tightly linked to previously reported QTLs regulating total number of spikelets per spike, number of sterile spikelets per spike and proportion of fertile spikelets per spike. Qsw-2D-1 was detected across stages, and was in the same marker region as a major QTL for plant height, QPH.cgb-2D.1. These indicate that these QTLs are on chromosomes 5A and 2D are involved in regulating these agronomic traits and are valuable for molecular breeding.