<p>Rice serves as a primary dietary source of calories for over half of the global population. Bacterial blight (BB), caused by the pathogen <i>Xanthomonas oryzae</i> pv<i>. oryzae</i> (<i>Xoo</i>), poses a significant challenge to rice cultivation worldwide, including Bangladesh. This study aimed to utilize a marker-assisted backcross breeding strategy to stack three BB resistance (R) genes (<i>Xa4</i>, <i>xa13</i>, and <i>Xa21</i>) into the genetic background of a susceptible mega rice variety BRRI dhan28. Because BRRI dhan28 inherently possesses the <i>Xa4</i> gene, which is ineffective when present alone against the prevalent BB isolates in Bangladesh. Donor parent IRBB60 was utilized as the source to introgress the <i>xa13</i> and <i>Xa21</i> genes, thereby achieving the three-gene stack. Backcrossing, selfing, and foreground selection were conducted across the generations (F₁ to BC₃F₇) to develop advanced resistant lines. Genotypic and phenotypic data analysis of 400 <span>BC</span>₃F₂ individuals revealed that BB resistance followed the simple Mendelian inheritance. Among the best selected 40 fixed BILs (backcross inbred lines) in the BC₃F₇ generation, 33 possessed all the candidate R genes, while four lines contained only <i>Xa4</i> and <i>Xa21</i>, and three lines consisted of <i>xa13</i> with <i>Xa4</i> genes. Disease scoring for BB ranged from 0 to 5 for these BILs, in contrast to 7 to 9 for the recipient parent. Marker-trait association analysis displayed a negative correlation between specific molecular markers and BB disease. The used molecular markers demonstrated significant potential in marker-assisted selection breeding. Furthermore, multi-location trials of the best eight BILs recorded that these lines possess broad-spectrum resistance to BB disease with superior agronomic performance. These BILs could serve as a promising candidate for developing novel rice cultivars resistance to bacterial blight or may be used as potential genetic stock for future rice breeding programs.</p>