Summary Broken and cracked eggshells contribute significantly to economic losses in the egg production industry. We previously identified ovocalyxin-32 as a potential gene influencing eggshell traits, by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation (DEF) conducted over 14 generations. We determined the nucleotide sequences of six ovocalyxin-32 exons in the parent individuals and analysed the association between ovocalyxin-32 and eggshell traits in the F(2) individuals. We identified three haplotypes (W, M and S) of ovocalyxin-32 in the parent individuals. A mismatch amplification mutation assay was performed to distinguish six diplotype individuals (WW, MM, SS, WM, MS and WS) in the F(2) population. The egg weight (EW) of SS-diplotype individuals was significantly higher than that of WW-, WM- and WS-diplotypes. Short length of the egg (SLE) of SS-diplotype individuals was significantly higher than that of WW-, WM- and MS-diplotypes. Long length of the egg (LLE) of SS-diplotype individuals was significantly higher than that of WM- and WS-diplotypes. DEF of WW-diplotype individuals was significantly higher than that of SS-, WM, MS and WM-diplotypes. Haplotypic effect analyses showed significant differences between the W-haplotype and the S-haplotypes in the EW, SLE, LLE and DEF. The DEF of M-haplotype was significantly lower than that of W- and S-haplotypes. These results suggest that S- and M-haplotypes are critical for high quality of eggshells in the F(2) population. In conclusion, ovocalyxin-32 is a useful marker of eggshell traits and can be used to develop strategies for improving eggshell traits in commercial layer houses.