ABSTRACT. Nucleotide-binding site (NBS) disease resistance genes play a crucial role in plant defense responses against pathogens and insect pests. Many NBS-encoding genes have been detected in Lotus japonicus, an important forage crop in many parts of the world. However, most NBS genes identified so far in L. japonicus were only partial sequences. We identified 45 full-length NBS-encoding genes in the L. japonicus genome, and analyzed gene duplications, motifs, and the molecular phylogeny to further understand the NBS gene family. We found that gene duplication events rarely occur in L. japonicus NBS-encoding (LjNBS) genes. In addition, LjNBS genes were subjected to selection pressure, and codon usage bias was evident. We tested for purifying selection (specifically in the CC-NBS-LRR and TIR-NBS-LRR groups), and found strong purifying selection in the TIR-domain-containing sequences, indicating that the CC-NBS-LRR group is more likely to undergo expansion than the TIR-NBS- LRR group. Moreover, our results showed that both selection and mutation contributed to LjNBS codon usage bias, but mutational bias was the major influence on codon usage.