The study aims to evaluate and compare the onset of local anesthesia (LA) and pain perception during endodontic treatment in hemophilic and thalassemic patients. The study included 90 patients with symptomatic irreversible pulpitis of the mandibular molars. Three groups ( = 30 in each group) were included. Group 1: hemophilic patients; group 2: thalassemic patients; and group 3: individuals without any systemic diseases. Onset of LA and visual analogue scale (VAS) scores was recorded immediately after the administration of local anesthesia, during the pulp exposure procedure, and during canal instrumentation, and were compared between the three groups. Frequency distribution, ANOVA, and linear regression analysis ( < 0.05) were applied. The mean onset time was 46 ± 34 s in the hemophilic group, 42 ± 23 s in the thalassemic group, and 38 ± 12 s in controls, but the differences were statistically insignificant. After LA administration (LA-VAS), all three groups experienced a statistically significant reduction in pain ( = 0.048). On pulp exposure (PE-VAS) ( = 0.82) and during canal instrumentation (CI-VAS) ( = 0.55), there was no statistically significant difference in pain perception between the groups. The coefficients indicate a positive correlation between the VAS and onset time, indicating a positive reduction in the VAS following the administration of LA. Hemophilic patients exhibited a clinically longer average onset time for LA. However, the difference among the three groups with regard to the overall pain perception after LA administration, during and after pulp exposure, and during canal instrumentation was statistically insignificant.