Barbels are important sensory organs in teleosts, reptiles, and amphibians. The majority of ?4,000 catfish species, such as the channel catfish (Ictalurus punctatus), possess abundant whisker-like barbels. However, barbel-less catfish, such as the bottlenose catfish (Ageneiosus marmoratus), do exist. Barbeled catfish and barbel-less catfish are ideal natural models for determination of the genomic basis for barbel development. In this work, we generated and annotated the genome sequences of the bottlenose catfish, conducted comparative and subtractive analyses using genome and transcriptome datasets, and identified differentially expressed genes during barbel regeneration. Here, we report that chemokine C-C motif ligand 33 (ccl33), as a key regulator of barbel development and regeneration. It is present in barbeled fish but absent in barbel-less fish. The ccl33 genes are differentially expressed during barbel regeneration in a timing concordant with the timing of barbel regeneration. Knockout of ccl33 genes in the zebrafish (Danio rerio) resulted in various phenotypes, including complete loss of barbels, reduced barbel sizes, and curly barbels, suggesting that ccl33 is a key regulator of barbel development. Expression analysis indicated that paralogs of the ccl33 gene have both shared and specific expression patterns, most notably expressed highly in various parts of the head, such as the eye, brain, and mouth areas, supporting its role for barbel development.