The preventive benefits of pit and fissure sealants rely on their retention and sealing ability. The drawback of the conventional pit and fissure sealant is its poor retention due to the presence of moisture. Among the numerous pit and fissure sealants available, newer hydrophilic sealants are ideal for children. This study evaluates and compares the sealing ability of hydrophilic sealant Embrace WetBond with conventionally used glass ionomer sealant under a stereomicroscope. A total of 48 extracted human premolars were randomly divided into two groups (N = 24) and sealed with Embrace WetBond and GC Fuji VII as per manufacturers' instructions. Following thermocycling, the sectioned samples were evaluated for sealant penetration, unfilled space, and total length of fissure under a stereomicroscope at magnifications 2.5×, 4×, and 5×. The values were measured in microns and in various fissure types using the "ImageJ app" to measure the sealant penetrability and sealing ability. The data recorded were statistically evaluated. The penetrability of moisture-tolerant sealant was better (87.8 ± 10.7) compared to that of glass ionomer sealant (73.8 ± 15.5) (p = 0.002). Among the samples, U-type fissure patterns displayed greater penetrability (94.2 ± 6.2), whereas IK-type fissures revealed the lowest degree of penetrability (67.5 ± 7.3). Embrace WetBond is better than glass ionomer sealant with respect to penetrability and sealing ability under stereomicroscope hence recommended as a better sealant for pediatric clinical practice. Priscilla S, Shivashankarappa PG, Mohandoss S, et al. Comparative Evaluation of Sealing Ability of Moisture-tolerant Sealant and Glass Ionomer Sealant Using Stereomicroscope: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(S-1):S67-S71.