Genetic diversity is vital for population survival, yet there is a paucity of studies focusing on the effectiveness of establishing protected areas for maintaining the population genetic diversity of threatened plant species. To evaluate the effectiveness of in situ conservation measures, we used simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) markers to monitor temporal dynamics in genetic diversity of the threatened wild rice Oryza rufipogon in both protected and unprotected populations in China between 2001 and 2020. Unprotected populations tended to have decreased census population sizes (Nc) compared to protected populations, although they both showed a reduction in effective population size (Ne). While allele diversity and expected heterozygosity remained stable, several populations, especially those without protection, exhibited a significant decrease in observed heterozygosity (Ho) and an increase in inbreeding (FIS). The level of genetic differentiation between populations did not change over time, but the number of private alleles increased and Ne varied in several populations, indicating the effects of genetic drift. Indicators for temporal trends in Ho, FIS, and Ne revealed that both protected and unprotected populations are facing warnings of declining genetic diversity, although some protected populations remain resilient, reflecting the genetic lag behind Nc change. Overall, these findings highlight the effectiveness of in situ conservation efforts in maintaining population size and genetic diversity, yet we also show the necessity of lasting population dynamics monitoring, using different genetic indicators.