The National Environmental Specimen Bank (NESB) has been sampling and cryogenically preserving various wildlife specimens to monitor bio-accumulations of chemical pollutants since 2010. Recently, the NESB set up a plan to develop reference materials at their facility to assure the analytical quality of and validate the analytical methods for their monitoring samples. One of the crucial characteristics of reference materials is intra- and inter-bottle homogeneity. In this study, we used ANOVA for total mercury concentrations in some samples to validate their homogeneities after milling and homogenization. We examined the intra- and inter-bottle homogeneities of two cryogenically-milled samples (Korean mussel (Mytilus coruscus) and black-tailed gull’s egg (Larus crassirostris). The variations in the total mercury concentrations were not significantly different intra- and inter-bottle (mussel: F=0.74, p=0.67; gull egg: F=1.96, p=0.10). Additionally, the relative standard deviations of the total mercury concentrations showed low values (mussel: 2.02%, gull egg: 1.78%). Therefore, the cryogenic-milling process statistically proves the homogeneity of the materials of mussels and sea gull eggs for chemical analysis for total mercury.