Accurate analysis of quantitative PET data plays a crucial role in studying small, specific brain structures. The integration of PET and MRI through an integrated PET/MR system presents an opportunity to leverage the benefits of precisely aligned structural MRI and molecular PET images in both spatial and temporal dimensions. However, in many clinical workflows, PET studies are often performed without the aid of individually matched structural MRI scans, primarily for the sake of convenience in the data collection and brain segmentation possesses. Currently, two commonly employed segmentation strategies for brain PET analysis are distinguished: methods with or without MRI registration and methods employing either atlas-based or individual-based algorithms. Moreover, the development of artificial intelligence (AI)-assisted methods for predicting brain segmentation holds promise but requires further validation of their efficiency and accuracy for clinical applications. This study aims to compare and evaluate the correlations, consistencies, and differences among the above-mentioned brain segmentation strategies in quantification of brain metabolism in 18F-FDG PET/MR analysis.Strong correlations were observed among all methods (r = 0.932 to 0.999, P < 0.001). The variances attributable to subject and brain region were higher than those caused by segmentation methods (P < 0.001). However, intraclass correlation coefficient (ICC)s between methods with or without MRI registration ranged from 0.924 to 0.975, while ICCs between methods with atlas- or individual-based algorithms ranged from 0.741 to 0.879. Brain regions exhibiting significant standardized uptake values (SUV) differences due to segmentation methods were the basal ganglia nuclei (maximum to 11.50 ± 4.67%), and various cerebral cortexes in temporal and occipital regions (maximum to 18.03 ± 5.52%). The AI-based method demonstrated high correlation (r = 0.998 and 0.999, P < 0.001) and ICC (0.998 and 0.997) with FreeSurfer, substantially reducing the time from 8.13 h to 57 s on per subject.Different segmentation methods may have impact on the calculation of brain metabolism in basal ganglia nuclei and specific cerebral cortexes. The AI-based approach offers improved efficiency and is recommended for its enhanced performance.© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.