ABSTRACT: MADS-box transcription factors (TFs) have fundamental roles in regulating floral organ formation and flowering time in flowering plants. In order to understand the function of MIKC-type MADS-box family genes in Cyclocarya paliurus (Batal.) Iljinskaja, we first implemented a genome-wide analysis of MIKC-type MADS-box genes in C. paliurus. Here, the phylogenetic relationships, chromosome location, conserved motif, gene structure, promoter region, and gene expression profile were analyzed. The results showed that 45 MIKC-type MADS-box were divided into 14 subfamilies: BS (3), AGL12 (1), AP3-PI (3), MIKC* (3), AGL15 (3), SVP (5), AGL17 (2), AG (3), TM8 (1), AGL6 (2), SEP (5), AP1-FUL (6), SOC1 (7), and FLC (1). The 43 MIKC-type MADS-box genes were distributed unevenly in 14 chromosomes, but two members were mapped on unanchored scaffolds. Gene structures were varied in the same gene family or subfamily, but conserved motifs shared similar distributions and sequences. The element analysis in promoters' regions revealed that MIKC-type MADS-box family genes were associated with light, phytohormone, and temperature responsiveness, which may play important roles in floral development and differentiation. The expression profile showed that most MIKC-type MADS-box genes were differentially expressed in six tissues (specifically expressed in floral buds), and the expression patterns were also visibly varied in the same subfamily. CpaF1st24796 and CpaF1st23405, belonging to AP3-PI and SEP subfamilies, exhibited the high expression levels in PA-M and PG-F, respectively, indicating their functions in presenting heterodichogamy. We further verified the MIKC-type MADS-box gene expression levels on the basis of transcriptome and qRT-PCR analysis. This study would provide a theoretical basis for classification, cloning, and regulation of flowering mechanism of MIKC-type MADS-box genes in C. paliurus.