Background: Forest regeneration is decisive for future forest development and therefore of major concern to forest ecologists. The ability of overstory tree species to regenerate successfully is important for the preservation of tree species diversity and its associated flora and fauna. This study investigated forest regeneration dynamics in the Cat Ba National Park, a biodiversity hotpot in Vietnam. Data was collected from 90 sample plots and 450 sub-sample plots in the regional limestone forests. We compared species richness between the regeneration and overstory tree layers and examined the effect of environmental factors on the occurrence of regeneration. We developed five ratios to relate overstory and regeneration richness and diversity. Results: We found 97 tree species in the regeneration layer compared to 136 species in the overstory layer. Average regeneration density was 3,764 ± 1,601 per ha. Around 70% of the overstory tree species generated offspring. Of the tree species threatened according to The International Union for Conservation of Nature’s Red List of Threatened Species, only 36% were found in the regeneration layer. A principal component analysis provided evidence that the regeneration of tree species was linked to terrain factors (percentage of rock surface, slope) and soil properties (Cation exchange capacity, pH, humus content, soil moisture, soil depth). Contrary to our expectations, the prevailing light conditions (total site factor, gap fraction, openness, indirect site factor, direct site factor) had no influence on regeneration density and composition, probably due to the small gradient in light availability. Conclusion: We conclude that tree species richness in Cat Ba National Park appears to be declining at present. We suggest similar investigations in other biodiversity hotspots to learn whether the observed trend is a global phenomenon. In any case, a conservation strategy for the threatened tree species in the Cat Ba National Park needs to be developed if tree species diversity is to be maintained.