Donkeys (Equus asinus) have played a vital role in agriculture, transportation, and companionship, particularly in developing regions where they are indispensable working animals. The domestication of donkeys marked a significant turning point in human history, as they became essential for transportation, agriculture, and trade, especially in arid and semi-arid areas where their resilience and endurance were highly valued. In modern society, donkeys are indispensable due to their diversified applications, including meat, dairy, medicine, and functional bioproducts, supporting economic, cultural, and medical industries. Despite their critical importance, research on donkeys has historically been overshadowed with studies on horses. However, recent advancements in high-throughput sequencing and bioinformatics have significantly deepened our understanding of the molecular landscape of donkey genome, uncovering their unique adaptations, genetic diversity, and potential therapeutic applications. Microsatellite and mitochondrial DNA (mtDNA) markers have proven effective in assessing the genetic diversity of donkeys across various regions of the world. Additionally, significant strides have been made in characterizing differentially abundant genes, proteins, and metabolic profiles in donkey milk, meat, and skin, and in identifying specific genes/proteins/metabolites associated with sperm quality, motility, and reproduction. Advanced genomic technologies, such as genome-wide association studies and the identification of selection signatures, have also been instrumental in delineating genomic regions associated with phenotypic and adaptive traits. This review integrates data from diverse studies, including those on genetic diversity, transcriptomics, whole genome sequencing, protein analysis, and metabolic profiling, to provide a comprehensive overview of donkey biology. It underscores the unique characteristics of donkeys and emphasizes the importance of continued research to improve their genetic management, conservation, and agricultural use, ensuring their ongoing contribution to human societies.