Nodulin-like (NL) genes are involved in transporting of various substances and may play key roles during the establishment of symbiosis in legumes plants. However, basic biological information of NL genes in the wheat genome is still largely unknown. Here, we identified and characterized NL genes in wheat via integrating genomic information, collinearity analysis, co-expression network analysis (WGCNA) and transcriptome analysis. In addition, we analyzed the polymorphisms and the roles of NL genes during arbuscular mycorrhizal (AM) symbiosis using a large wheat panel consists of 259 wheat genotypes. We identified 181 NL genes in the wheat genome, which were classified into SWEET, Early Nodulin-Like (ENODL), Major Facilitator Superfamily-Nodulin (MFS), Vacuolar Iron Transporter (VIT) and Early nodulin 93 (ENOD93) subfamily. The expansion of NL genes was mainly driven by segmental duplication. The bHLH genes are potential unrecognized transcription factors regulating NL genes. Moreover, two NL genes were more sensitive than other NL genes to AM colonization. The polymorphisms of NL genes are mainly due to random drift, and the natural mutation of NL genes led to significant differences in the mycorrhizal dependence of wheat in phosphorus uptake. The results concluded that NL genes potentially play important roles during AM symbiosis with wheat.