| Summary: | In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in β-carotene content. Thus, we aimed to incorporate the <i>crtRB1</i> gene from UMI285β<sup>+</sup> into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the β-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC<sub>1</sub>F<sub>1</sub> and BC<sub>2</sub>F<sub>1</sub> plants possessing the heterozygous allele for <i>crtRB1</i> and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC<sub>2</sub>F<sub>2</sub> seeds. In the BC<sub>2</sub>F<sub>2</sub> generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous <i>crtRB1</i>-favorable allele with maximum RPGR (86.74–90.16%) were selected and advanced to BC<sub>2</sub>F<sub>3</sub>. The four selected plants were selfed to produce BC<sub>2</sub>F<sub>3</sub> and then evaluated for agronomic traits and β-carotene content. The agronomic performance of the four lines was similar (78.83–99.44%) to that of the recurrent parent, and β-carotene content (7.541–8.711 μg/g) was on par with the donor parent. Our study is the first to improve the β-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.
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