The wild relatives of cultivated rice offer crucial resistance genes for combating stresses like drought. Developing rice varieties with drought tolerance is possible through chromosome segment substitution lines (CSSLs), which blend the genetic background of a high-yielding parent with specific chromosome segments from a donor parent. This study aimed to study the effect of drought stress on various root traits of chromosome segment substitution lines (CSSLs) and their relationship with specific molecular markers. Ninety-six genotypes, including 80 chromosome segment substitution lines (Curinga x O. rufipogon and Curinga x O. meridionalis), 9 New Rice for Africa (NERICAs) and 7 controls were grown in Basket and PVC pipe methods for phenotyping different root traits. Under drought stress (DS), MER16, MER20, RUF10, RUF16, RUF44, NERICA1, and NERICA3 showed superior performance for most of the root traits. These evaluations were supplemented with association analysis of 17 root trait-linked simple sequence repeat (SSR) markers with root phenotypic traits. The marker RM201 is strongly associated with multiple root traits, found to be independent of three growth conditions (well-watered “WW” under Basket, WW condition and DS conditions under PVC pipe). The marker RM316 is associated with root volume, and the marker RM7424 and RM1054 show maximum root length. In conclusion, these markers can be used in marker-assisted breeding programs, and the lines carrying them can be used as parental lines in variety-development programs for drought tolerance.