In order to preliminarily explore the causes of eggshell dark spots, the thickness, hardness, moisture and protein content of calcareous hard eggshell, and the thickness and protein content of shell membrane were measured in the normal eggshell and dark spots portion as well as no spots portion of dark spots eggshell, and the ultra structure was also studied results: The hardness of calcareous hard eggshell of dark spots eggshell was minimum, and its water content was maximum, which were highly significant (P<0.01) compared with other groups; However, there with no difference between spots group and the normal group. The thickness of calcareous hard shell was similar among the three groups, while that of shell membrane was highly significant (P < 0.05). In addition, protein content of calcareous hard shell of three groups had no significant difference (P > 0.05), while that of shell membrane of dark spots egg was much lower than the normal egg (P < 0.01). The result of scanning electron microscope showed that both the calcareous hard shell and shell membrane of dark spots group had more significant loose texture and porosity features than other groups. In conclusion, the formation of dark spots was mainly due to that the shell membrane under eggshell dark spots was thin with large mesh, resulting in moisture of the egg permeated through the shell membrane into the calcareous hard shell easily, and then the moisture was absorbed by calcareous hard shell, resulting in the formation of dark spots gradually.