Collagen XXVIII is the last discovered member of the collagen superfamily and thus has been only sparsely investigated. We studied collagen XXVIII in zebrafish to gain insight into its structure, evolution and expression. In contrast to human and mouse, the zebrafish genome contains four collagen XXVIII genes, col28a1a and -b, and col28a2a and -b. Genomic context and phylogenetic analysis revealed that the a2 branch was lost during evolution of mammals, whereas the duplication of the a1 and a2 branches results from the whole genome duplication in the teleost lineage. Sequence analysis revealed conservation of domain structure and the unique imperfections in the triple helical domain. Two major forms of collagen XXVIII were identified, Col28a1b in adult and Col28a2a in 3-5dpf zebrafish. Composite agarose/polyacrylamide gel electrophoresis revealed that both these chains mainly form dimers of trimers, although Col28a1b appears to be more polydisperse. Homodimers are abundant, although it is possible that complexes consisting of Col28a2a and Col28a1a or -a2b occur. Peptide mass fingerprint analysis revealed that the C-terminal Kunitz domain is often proteolytically processed. In contrast to murine collagen XXVIII, the zebrafish orthologs are widely expressed and not only present in the nervous system. They are differentially expressed in the liver, thymus, muscle, intestine and skin. Altogether our results point to a unique nature of collagen XXVIII within the collagen family.