MIKC-type MADS-box transcription factors constitute one of the largest gene families in plants, playing pivotal roles in regulating plant growth and development, hormone signaling transduction, and responses to biotic and abiotic stresses. However, there have been no reports on the systematic identification and characterization of MIKC-type MADS-box proteins in walnuts. In this study, we identified 52 JrMADS genes in the walnut genome and transcriptome, and categorized them into 14 subfamilies through structural domain and phylogenetic tree analysis. It was found that these genes were unevenly distributed across 16 chromosomes. Within the MIKC-type MADS-box gene family, we identified three pairs of tandem-duplicated genes and 40 pairs of segmental duplicated genes, indicating that segmental duplication was the primary mechanism of gene amplification in walnut. Ka/Ks analysis showed that the family genes have undergone purifying selection during evolutionary processes. The promoter was predicted to contain cis-acting elements related to growth, development, plant hormones, and stress response. Expression profile analysis showed that JrMADS genes have different expression patterns in various tissues and developmental stages of male and female flower buds. Notably, an ancient clade of TM8 (JrMADS43) genes was found, which is absent in Arabidopsis but present in other flowering plants. Another gene, TM6 gene (JrMADS4), belongs to the AP3 subfamily and is a clade that has diverged from tomatoes. Through qPCR analysis, we verified the differential expression of JrMADS genes at different developmental stages (MB-1/2/3 and FB-1/2/3), with JrMADS5, JrMADS8, JrMADS14, JrMADS24, JrMADS40, JrMADS46, JrMADS47, JrGA3ox1, and JrGA3ox3 showing significantly higher expression in male than in female flower buds. In summary, our results provide valuable information for further biological functions research on MIKC-type MADS-box genes in walnut, such as flower organ development, and lays a solid foundation for future studies.