The reproductive cycle of plants features a crucial transition between diploid sporophytic and haploid gametophytic generations. In garlic ( Allium sativum L .), a lack of gametophyte fertility poses significant challenges for breeding. This study conducted a comprehensive comparative transcriptomic analysis across three developmental stages of garlic floral buds from three genotypes with varied fertility profiles to unravel the genetic underpinnings of gametophyte development. Through differential expression analysis and weighted gene co-expression network analysis (WGCNA), we identified key pathways and genes influencing gametophyte fertility. Our analysis revealed significant enrichment in pathways related to lipid metabolism, amino acid biosynthesis, nucleic acid metabolism, and ribosome biogenesis, which are pivotal for gametophyte vitality and development. Furthermore, we identified the AsAMS gene as a key regulator of gametophyte fertility, that may orchestrat tapetal development and microspore formation by modulating the expression of genes involved in lipid biosynthesis and transport, thereby playing a crucial role in pollen viability. The first functional validation using virus-induced gene silencing (VIGS) in garlic further substantiated the role of AsAMS, which demonstrated its critical impact on pollen viability and morphological integrity of reproductive structures. Taken together, these findings not only deepen our understanding of the genetic mechanisms underlying gametophyte development in garlic but also shed light on potential genetic interventions to overcome fertility barriers. By delineating the pathways and key regulators such as AsAMS, this study opens new avenues for enhancing reproductive efficiency in garlic.