Purpose: Brain PET/CT examinations are generally performed using a 1 Bed acquisition. With the increasing use of PET/CT systems with an extended axial field of view (AFOV), the overlap region between adjacent Bed positions can be utilized for image acquisition. This study aimed to evaluate differences in relative sensitivity, image quality, and quantitative characteristics between 1 Bed and 2 Bed overlap acquisitions using a PET/CT system with a 30 ㎝ AFOV and to evaluate the applicability of 2 Bed overlap acquisition for Brain PET/CT examinations. Materials and Methods: Images were acquired using a GE Discovery MI Gen2 PET/CT scanner (GE HealthCare, Milwaukee, WI, USA) with a 30 ㎝ AFOV. For the evaluation of relative sensitivity, a Custom-made Phantom and a PET Annulus Phantom were used for 1 Bed and 2 Bed overlap acquisitions, and relative sensitivity was compared over the same axial image range. For image quality and quantitative evaluation, a Hoffman 3D Brain Phantom was scanned under three acquisition conditions: 1 Bed center, 1 Bed edge, and 2 Bed overlap. Image quality and quantitative characteristics were evaluated using the signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), percent coefficient of variation (%COV), and mean standardized uptake value (SUV_mean). Results: The relative sensitivity of the 2 Bed overlap acquisition was slightly higher than that of the 1 Bed acquisition, with mean values 2.0% higher (range, 0.1–5.8%) for the Custom-made Phantom and 1.2% higher (range, 0.1–2.0%) for the PET Annulus Phantom. In the Hoffman 3D Brain Phantom, the 2 Bed overlap acquisition showed more favorable image quality metrics than the 1 Bed acquisitions. Compared with the 1 Bed center and edge acquisitions, respectively, SNR was higher by 3.9–15.2% and 19.8–35.0%, CNR was higher by 3.4–21.0% and 8.5–37.0%, and %COV was lower by 3.7–13.2% and 16.4–25.9%. In the quantitative evaluation, SUV_mean values in the frontal and occipital lobes were generally similar across the three acquisition conditions. Conclusion: The difference in relative sensitivity between the 1 Bed and 2 Bed overlap acquisitions was small. However, the 2 Bed overlap acquisition showed higher SNR and CNR and lower %COV than the 1 Bed acquisitions. SUV_mean values in the frontal and occipital lobes were generally similar across the three acquisition conditions. These findings suggest that 2 Bed overlap acquisition may be considered as an alternative acquisition strategy for Brain PET/CT using a 30 ㎝ AFOV PET/CT system. Further studies using standardized NEMA NU 2 sensitivity measurements and clinical data are warranted to evaluate the clinical applicability of this acquisition strategy.