Drought, acidity, and P deficiency are problems on upland rice production in Ultisol soil in Indonesia. Pup1 locus in rice plant has a role in uptaking P nutrient and is suspected to function for tolerant to drought. Some BC2F6 lines derived from Situ Bagendit varietal cross containing Pup1 locus had been developed through Markerassisted Backcrossing method, for P uptake and for resistance to blast. This research was aimed to evaluate the effect of Pup1 locus to drought stress condition. The study was divided into two experiments, namely test of polyethylene glycol (PEG) 8000 and root penetration ability test (DTA). The experiment was conducted at the Greenhouse of Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, from November 2012 until May 2013. The number of lines was 52 lines, consisted of 24 lines of BC2F6 Situ Bagendit × Kasalath (= SK), 22 lines of Situ Bagendit × NIL-C433 (= SN), three parents namely Situ Bagendit (recurrent parent), Kasalath, and NIL-C433, two tolerant controls for drought namely Cabacu and Gajah Mungkur, and one sensitive control for drought IR20. The research used a completely randomized design for test PEG 8000 and a split-split plot design for test of root penetration ability. Results showed that maximum PEG 8000 concentrations for drought stress study on rice were 20%. Plant containing Pup1 locus was more tolerant to drought stress in PEG 8000 experiment through its longer roots and plumules. Pup1 locus gave a positive effect on root penetration ability, with longer roots and more able to penetrate wax layer. Plant carrying Pup1 locus also showed tolerant to drought on Ultisols soil, as indicated by the increase of dry weight of root and shoot compare to those of parents without Pup1 locus.