Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes (79.60 ± 0.77 μm vs. 101.46 ± 1.07 μm, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found (79.51 ± 2.36 μm vs. 101.46 ± 1.07 μm, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured (1.48 ± 0.42 μm) than in vitro matured for 72 and immature oocytes (1.10 ± 0.16 and 0.43 ± 0.12 μm, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.