, a plant-specific gene family, encode proteins containing domain of unknown function (DUF) domains. Despite their essential functions, there is a lack of insight into TaDUF668s. Here, 31 were identified from the wheat genome; according to phylogenetic relationships, they were named to . All TaDUF668s were hydrophilic and unstable proteins. There were 22 TaDUF668s that showed subcellular localization in nucleus. Evolutionary analysis demonstrated that TaDUF668s had undergone strong purifying selection, and fragment duplication plays major role in TaDUF668 family expansion. -element prediction displayed that over 90% of TaDUF668 promoter regions contain the growth and abiotic responsiveness element. Consistently, expression profiling showed that were highly induced in five wheat growth and development stages, seven main different tissues, five abiotic stresses, and five pathogenic stresses. In total, 12 were targeted by 20 miRNAs through the inhibition of translation and cleavage patterns. RT-qPCR results confirmed that the expression of six was significantly regulated by NaCl, PEG, , and ; nevertheless, the regulation patterns were different. In summary, through systematic identification, characterization, evolutionary analysis, and expression profiling, a comprehensive understanding of has been obtained, which lays a foundation for further functional studies of .