Aquaporins (AQPs), a type of intrinsic membrane proteins that transport water and small solutes across biological membranes, play crucial roles in plant growth and development. This study presents a first genome-wide identification and comparative analysis of the gene family in papaya ( L.), an economically and nutritionally important fruit tree of tropical and subtropical regions. A total of 29 genes were identified, which represent five subfamilies, i.e., nine plasma intrinsic membrane proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), seven NOD26-like intrinsic proteins (NIPs), two X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). Although the family is smaller than the 35 members reported in Arabidopsis, it is highly diverse, and the presence of genes as well as orthologs in and implies that the complete loss of the XIP subfamily in Arabidopsis is lineage-specific, sometime after its split with papaya but before Brassicaceae-Cleomaceae divergence. Reciprocal best hit-based sequence comparison of 530 AQPs and synteny analyses revealed that genes belong to 29 out of 61 identified orthogroups, and lineage-specific evolution was frequently observed in Brassicales. Significantly, the well-characterized NIP3 group was completely lost; lineage-specific loss of the NIP8 group in Brassicaceae occurred sometime before the divergence with Cleomaceae, and lineage-specific loss of NIP2 and SIP3 groups in Brassicaceae occurred sometime after the split with Cleomaceae. In contrast to a predominant role of recent whole-genome duplications (WGDs) on the family expansion in , , and Brassicaceae plants, no recent repeats were identified in papaya, and ancient WGD repeats are mainly confined to the PIP subfamily. Subfamily even group-specific evolution was uncovered via comparing exon-intron structures, conserved motifs, the aromatic/arginine selectivity filter, and gene expression profiles. Moreover, down-regulation during fruit ripening and expression divergence of duplicated genes were frequently observed in papaya. These findings will not only improve our knowledge on lineage-specific family evolution in Brassicales, but also provide valuable information for further studies of genes in papaya and species beyond.