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Abstract Background and purposes Brain morphometry is an important assessment technique to assess certain morphological brain features of various brain regions, which can be quantified in vivo by using high-resolution structural magnetic resonance (MR) imaging. This study aims to investigate the effect of different types of pulse sequence on regional corpus callosum (CC) morphometry analysis. Materials and methods Twenty-one healthy volunteers were scanned twice on the same 3T MRI scanner (Magnetom Trio, Siemens, Erlangen, Germany) equipped with an 8-channel head coil. Two different MR pulse sequences were applied to acquire high-resolution 3D T1-weighted images: magnetization-prepared rapid gradient-echo (MP-RAGE) and modified driven equilibrium Fourier transform (MDEFT) pulse sequence. Image quality measurements such as SNR, contrast-to-noise ratio, and relative contrast were calculated for each pulse sequence images independently. The values of corpus callosum volume were calculated based on the vertex of reconstructed surfaces. The paired dependent t test was applied to compare the means of two matched groups. Results Three sub-regional CC, namely anterior, mid-anterior, and posterior, resulted in an estimated volume difference between MDEFT and MP-RAGE pulse sequences. Central and mid-posterior sub-regional CC volume resulted in not significant difference between the two named pulse sequences. Conclusion The findings of this study demonstrate that combining data from different pulse sequences in a multisite study could make some variations in the results.