Three-dimensional (3D) structures of β-microglobulin (β2m) from chicken and various mammals have been described previously, but aside from genomic sequences, very little is known about the 3D structures of β2m in species other than warm-blooded vertebrates. Here, we present the first 3D structure of β2m from bony fish grass carp (Ctid-β2m), resolved at 2.1 Å. The key structural differences between this new structure and previously published structures are two new hydrogen bonds at positions 37-Ile and 38-Glu in strand C and 66-Lys in strand E, and a hydrophobic pocket around the centre of the protein found in Ctid-β2m. Importantly, Ctid-β2m has a short D strand and a long loop between stands C and D, rather than the flexible region found in other β2m structures that serves as a putative binding region for the major histocompatibility complex heavy chain. Comparing the Ctid-β2m structure with those of bovine and human β2ms, the Cα r.m.s.d of the latter are 1.3Å and 1.8Å respectively. Compared with the constant domains of Lamprey TCR-like receptor (Lamp-TCRLC) and Amphioxus V and C domain-bearing protein (Amphi-VCPC), Ctid-β2m exhibits very different topology. The 3D structures of domains predicted from Amphi-VCPC/Lamp-TCRLC are distinctly lacking in strand A of β2ms. There are 18 amino acids at the N-terminus of Amphi-VCPC that may have evolved into strand A of β2ms. A mutation in the BC loops of Amphi-VCPC may have led to the novel topology found in β2m. Based on these results, Ctid-β2m may well reflect evolutionary characteristics of ancestral C-set molecules.