Abstract: Background: Male sterile line regulated by temperature is called Thermo Sensitive Genetic Male Sterile (TGMS) line. The TGMS line can be reproduced under temperature condition below the critical temperature. Objective: This study aims to develop TGMS lines of rice using Marker Assisted Backcrossing (MAB). Methodology: One TGMS line, ANT 2-5-2-4-4, was used as donor parent. This line has tmsX gene which is located on rice chromosome 2. The SSR markers were applied in each backcross generation for assisting selection of rice plants having tmsX gene. Results: Two of the 32 lines were selected from BC2F1 generation. Both of them were identified as TGMS lines with highest genetic background. They had 97.22% genetic background of their recurrent parent based on 36 SSR markers which are located across all the chromosomes of rice. Conclusion: They will be further utilized as female parent for two-line hybrid rice production.