Natural regeneration in tropical forests is considered an essential part of forest restoration efforts; however, it is often under-estimated where the main focus has traditionally been on tree planting. This study assessed natural regeneration and its potential for the conservation of native tree species in Sitapahar Forest Reserve, Bangladesh. We established 99 temporary plots (2 m × 2 m each) in three canopy classes, namely dense canopy (DC, 70–100% canopy coverage), moderate canopy (MC, 40–70%), and open canopy (OC, <40%). In each plot, regenerating tree species were counted, identified, and height (m), collar diameter (cm), and diameter at breast height (dbh, cm) were measured. We identified 79 regenerating tree species under 31 plant families of which 61 were found in DC areas followed by 56 and 36 in MC and OC areas, respectively. Most of the species in DC areas were late-successional, while the OC areas were dominated by early successional species. Diversity and density of regeneration were lowest in OC areas. Findings of this study suggest harnessing biodiversity conservation by promoting natural regeneration and identifying the areas with higher potential for assisted natural regeneration that will enable in situ conservation of rare and vulnerable species by protecting them from further erosion.