This study aimed to compare the volume and quality of the newly formed bone following application of two types of xenografts and one synthetic material in bone defects in rabbit calvaria from histological and micro-CT aspects. Four 8-mm defects were created in 12 rabbit calvaria. Three defects were filled with bone substitutes and one was left unfilled as the control group. The newly formed bone was evaluated histologically and also by micro-CT at 8 and 12 weeks after the intervention. The percentage of osteogenesis was comparable in histomor-phometric assessment and micro-CT. Histological analysis showed that the percentage of the newly formed bone was 10.92 ± 5.17%, 14.70 ± 11.02%, 11.47 ± 7.04%, and 9.45 ± 5.18% in groups bovine 1, bovine 2, synthetic, and negative control, respectively after 8 weeks. These values were 33.70 ± 11.48%, 26.30 ± 18.05%, 22.92 ± 6.30%, and 14.82 ± 8.59%, respectively at 12 weeks. The difference in the percentage of the new bone formation at 8 and 12 weeks was not significant in any group (<i>P</i> > 0.05) except for bovine 1 group (<i>P</i> < 0.05). Micro-CT confirmed new bone formation in all groups but according to the micro-CT results, the difference between the control and other groups was significant in this respect (<i>P</i> < 0.05). All bone substitutes enhanced new bone formation compared with the control group. Micro-CT assessment yielded more accurate and different results compared with histological assessment.