Aquaporin proteins are part of an ancient family that functions as water transporting facilitators in all organisms. Phylogenetic and physiological analyses have revealed that plant aquaporins consist of two groups: the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs). Using the conserved asparagineproline-alanine (NPA) to NPA motif regions, we studied the evolution of 35 plant aquaporins that included nine of our newly cloned cucumber aquaporins and 26 from the GenBank database. Results indicated that NPA repeated regions were effective for phylogenetically characterizing the plant aquaporin family, and to accurately localize the introns. Phylogenetic analysis showed that 35 plant aquaporins fell into two distinct groups (except for the Arabidopsis gene AtMip)—PIPs and TIPs. The nine cucumber aquaporins belong to the PIP group that were localized further into two different sub-groups. The intron analysis showed that introns of plant aquaporins mainly consist of two types. Eighteen PIPs shared identical intron positions localized in connecting loop C between amino acids 95 and 96. Nine TIPs shared the other identical intron positions localized in connecting loop D between amino acids 44 and 45. Cucumber aquaporins CRB9 and CRB10 (with no intron in the repeated NPA regions) may be the result of intron loss events, while intronless rice (Orzya sativa) Os-TIP1 and Os-TIP2 may have resulted from other intron loss events. PIP11 and Os-PIP do not have the same amino acid number as major PIP members, but combined phylogenetic analysis results along with intron positions and phases showed that they belong to the PIP group. The phylogenetic tree and intron position information suggest that AtMip was mis-annotated as a member of aquaporin, and is a homologue of the glycerol facilitator-like protein. Introns share identical positions and phases within the PIP group (except PIP13) or the TIP group, but differ between the plasma and the tonoplast membrane aquaporins matching the phylogenetic analysis results. Intron positions of the repeated NPA regions of plant aquaporins that have stable inheritance can act as molecular markers for phylogenetic studies.