Brassica juncea, a member of the Brassicaceae family commonly referred to as mustard, is an allotetraploid (AABB, 2n=36) resulting from interspecific hybridization between Brassica rapa (AA, 2n=20) and Brassica nigra (BB, 2n=16). In this study, microspores were cultured using F₁ hybrids between two selected B. juncea double haploid (DH) lines with high anthocyanin and glucosinolate contents in the maternal versus leafy paternity. The results of the microspore culture showed that the total number of cultured buds, obtained embryos, and embryos per bud were 2,010, 15,526, and 7.62, respectively. Colchicine was used to induce the amphidiploids (AABB). We found that 149 of the 218 regenerated plants (68.3%) were amphidoploid. Among them, 117 individuals underwent ripening and seed harvesting and were used for subsequent phenotype analysis. Leaf color, length, and other agricultural traits exhibited various patterns owing to the recombination of the parental allele. The glucosinolate (GSL) content ranged from a maximum of 29.27 μmol/g dry weight to a minimum of 6.39 μmol/g dry weight, exceeding the range of parental value. The composition of GSL was mainly aliphatic, with sinigrin accounting for approximately 89% of the total aliphatic GSL content. These DH lines and their respective unique phenotypic traits are crucial for crop breeding and are valuable resources for advancing functional genomics and developing molecular markers in B. juncea.