Nowadays, new sealing materials have been marketed. Little is published about the performance of new materials. Objective: The purpose of this in vitro study was to determine the effect of fissure morphology and two different sealing materials on the microleakage and penetration ability of fissure sealants. Methods: Twenty extracted human molar teeth were randomly divided into two groups of 10 each. All teeth were cleaned with pumice prophylaxis followed by etching 60 seconds with 35% phosphoric acid, rinsing and drying with oil-free air. Two commercial, light cure sealants (Concise and Clinpro, 3M Dental USA) were applied following manufacturers' instructions. Each tooth was subjected to thermal cycling (5000 cycles at 5-55°C) and dye immersion (5% methylene blue for 24 hours). After sectioning, microleakage, penetration ability (unfilled area in mm2) and fissure morphology were examined. Fissure morphology was categorized as follows: U type, V type, Y1 and Y2 type. Multiple regression analyses were used for analysis. Results: the Clinpro group exhibited slightly less microleakage and less unfilled area compared to the Concise group. However, there were no statistically significant differences in microleakage and penetration ability between two different sealing materials. The significant impact on penetration ability was the fissure morphology (p<0.001). Conclusion: The different sealing materials had no effect on microleakage and penetration ability. Fissure morphology was significantly related to the penetration ability of sealants.