F-Box proteins act as versatile substrate adapters in SCF ubiquitin ligase complexes, regulating targeted proteolysis to control eukaryotic signaling, including cell cycle and stress responses. This study comprehensively characterizes the F-Box gene family in water buffalo (Bubalus bubalis), identifying 70 genes across 23 autosomes and the X chromosome via HMMER and BLASTP against Pfam domains. The results of phylogenetic analysis with orthologs from Homo sapiens, Mus musculus, Bos taurus, and Bubalus bubalis classified them into four subfamilies (FBX, FBXL, FBXW, FBXO), with 58 core buffalo genes forming a cohesive cluster. MEME analysis revealed nine conserved motifs, including the F-Box domain (motifs 1–2, e.g., LPDELLLYIFSYLDA), LRR for specificity (motif 3), and WD40 repeats for scaffolding (motifs 5–7,9), validated by Pfam. Gene structures varied from 2 to 23 exons, reflecting regulatory diversity. Synteny with Bos taurus showed ~85% ortholog conservation, indicating artiodactyl heritage. Physicochemical properties included MW 18.10–133.50 kDa, pI 4.27–10.41, and negative GRAVY (-0.75 to 0.143), suggesting hydrophilic profiles. Five segmental duplications (e.g., FBXO25/FBXO32) had Ka/Ks <1 (0.144–1.194), implying a purifying selection and divergence of 147–666 MYA via Ks Poisson correction. nsSNP analysis in 20 genes using PredictSNP, PolyPhen-2, and SIFT indicated 95% benign/synonymous effects, with exceptions like FBXO16 V160T potentially impairing substrate binding. WoLF PSORT predicted 28 nuclear, 12 cytoplasmic, 11 plasma membrane, 6 mitochondrial, and 4 extracellular and dual localizations, suggesting roles in transcription, degradation, and trafficking. This atlas elucidates F-Box evolution in ruminants and nominates CRISPR targets to enhance buffalo resilience to stressors in tropical agriculture.