BackgroundAssessment of segregating populations for their ability to withstand drought stress conditions is one of the best approaches to develop breeding lines and drought tolerant varieties. Bambara groundnut (Vigna subterraneaL. Verdc.) is a leguminous crop, capable of growing in low-input agricultural systems in semi-arid areas. An F4bi-parental segregating population obtained from S19-3 × DodR was developed to evaluate the effect of drought stress on photosynthetic parameters and identify QTLs associated with these traits under drought-stressed and well-watered conditions in a rainout shelter.ResultsStomatal conductance (gs), photosynthesis rate (A), transpiration rate (E) and intracellular CO2(Ci) were significantly reduced (p< 0.05) while water use efficiency (WUE) was significantly increased (p< 0.05) under drought-stressed conditions. A strong linear correlation was observed betweengs,WUE,A, EandCiunder both water regimes. The variability between different water treatment, among individual lines and the interaction between lines and environment for photosynthetic parameters provides resources for superior lines selection and drought resistant variety improvement. Significant QTL forgsandFV/FMunder well-watered conditions were mapped on LG5 and LG3, respectively, with more than 20% of the PVE, which could be considered as the major QTL to control these traits. Five clustered QTLs for photosynthetic traits under drought-stressed and well-watered conditions were mapped on LG5, LG6A, LG10 and LG11, respectively.ConclusionsSignificant and putative QTLs associated with photosynthetic parameters and the effect of drought stress on these traits have been revealed by QTL linkage mapping and field experiment in the F4segregating population derived from S19-3 × DodR in bambara groundnut. The study provides fundamental knowledge of how photosynthetic traits response to drought stress and how genetic features control these traits under drought-stressed and well-watered conditions in bambara groundnut.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04293-w.