Pineapple (Ananas comosus) ranks as the world's third most important tropical fruit and is a significant economic crop in China's tropical regions. To investigate the role of the dihydroflavonol 4-reductase (DFR) gene family in anthocyanin accumulation in pineapple and provide a theoretical foundation for germplasm improvement, this study identified and preliminary functionally analyzed members of the pineapple DFR gene family. A total of 17 dihydroflavonol 4-reductase genes were identified in the whole genome of pineapple, which were named AcDFR1 to AcDFR17 and distributed unevenly on 10 chromosomes. AcDFRs encoded proteins ranging from 313 to 378 amino acids (aa) in length, with molecular weights between 34.73 and 47.74 kDa and isoelectric points (pI) from 5.10 to 8.9. The secondary structure of AcDFR proteins primarily consisted of α-helices and β-sheets, with α-helix content ranging from 38.73% to 43.07% and β-sheet content from 13.24% to 15.64%. Phylogenetic analysis revealed that the 17 AcDFRs formed four subfamilies. The motif arrangement and number of exons were similar among AcDFRs. The AcDFR genes promoter regions contained multiple cis-acting elements responsive to light, hormones, abiotic stress, growth and development. Real-time quantitative PCR analysis revealed significant expression variations of AcDFRs genes across different pineapple tissues (roots, stems, leaves, flowers, pulp, peel), though most genes exhibited high expression in both pulp and peel. Cloning and identification of AcDFRs revealed sequences largely consistent with predicted sequences. Subcellular localization studies of the AcDFR1 in tobacco leaves via transient expression indicated localization to the cytoplasm and cell membrane. The findings would provide theoretical references for further investigation into the functions of the dihydroflavonol-4-reductase gene family in pineapple and for pineapple quality improvement.