Simple SummaryBroilers provide nutrition for people all around the world, and their muscle and fat contribute major roles. However, little is known about the relationships between the kruppel-like factor (KLF) and the development of muscle and fat. In recent years, studies have shown thatKLFs are involved in many physiological processes such as cell development, adipocyte differentiation and neurodevelopment. In our study, we aim to learn the functionsKLFs play in the muscle and fat development of chickens. Various bioinformatics analyses were used to illustrate the genomic information ofKLFs. A qPCR was performed to show the relative expression level ofKLFs. In addition, we collected RNA-seq data and we found thatKLFs have different expression levels in the same tissues at different points in time. This study helps explore the regulation mechanism ofKLFs in skeletal muscle and fat, and provides a theoretical basis for broiler breeding.AbstractThe kruppel-like factor (KLF) gene family is a group of transcription factors containing highly conserved zinc-finger motifs, which play a crucial role in cell proliferation and differentiation. Chicken has been widely used as a model animal for analyzing gene function, however, little is known about the function of theKLFgene family in chickens. In this study, we performed genome-wide studies of chickenKLFgenes and analyzed their biological and expression characteristics. We identified 13KLFgenes from chickens. Our phylogenetic, motif, and conserved domain analyses indicate that theKLFgene family has remained conserved through evolution. Synteny analysis showed the collinear relationship amongKLFs, which indicated that they had related biomolecular functions. Interaction network analysis revealed thatKLFs worked with 20 genes in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed thatKLF2was involved in Apelin and Forkhead Box O (FOXO) signaling pathways. Moreover, qPCR showed that 13KLFgenes were expressed in the nine selected tissues and displayed various gene expression patterns in chickens. RNA-seq showed thatKLF3andKLF10genes were differentially expressed in the normal and high-fat diet fed groups, andKLF4,KLF5,KLF6,KLF7,KLF9,KLF12, andKLF13genes were differentially expressed between undifferentiated and differentiated chicken preadipocytes. Besides, RNA-seq also showed thatKLFgenes displayed different expression patterns in muscle at 11 and 16 embryonic days old, and in 1-day-old chickens. These results indicated that theKLFgenes were involved in the development of muscle and fat in chickens. Our findings provide some valuable reference points for the subsequent study of the function ofKLFgenes.