ABSTRACT Background: Eggshell ultrastructure organization, including effective layer thickness, mammillary layer thickness, and average size of mammillary cones, is important for breeding and significantly influences eggshell mechanical properties. Several matrix proteins were known to be important in eggshell formation. However, the proteins and variations that determine eggshell ultrastructure organization are not known. Results: In this study, 17 single-nucleotide polymorphisms of three major genes in a hen population using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Five single-nucleotide polymorphisms with a very low minor allele frequency (< 1%) were excluded from further analysis. The remaining 12 single-nucleotide polymorphisms in Hardy-Weinberg equilibrium were used for analysis of associations with eggshell ultrastructure organization. Associations were found for (i) ovocleidin-116 with effective layer thickness (EFF), mammillary layer thickness (MAM), and average size of mammillary cones (SMAM); (ii) ovalbumin with eggshell thickness (ESH), effective layer thickness, and density of the mammillary cone (DMAM); and (iii) calmodulin1 with density of the mammillary cone. Conclusions: The single-nucleotide polymorphisms identified in the present study may be used as potential markers to improve eggshell quality.