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Background: Countries within the Greater Mekong Sub-region (GMS) of Southeast Asia have committed to eliminating malaria by 2030. Although the malaria situation has greatly improved, malaria transmission remains at international border regions. In some areas, Plasmodium vivax has become the predominant parasite. To gain a better understanding of transmission dynamics, knowledge on the changes of P. vivax populations after the scale-up of control interventions will guide more effective targeted control efforts. Methods: This study investigated genetic diversity and population structures in 206 P. vivax clinical samples collected at two time points in two international border areas: the China-Myanmar border (CMB) (n = 50 in 2004 and n = 52 in 2016) and Thailand-Myanmar border (TMB) (n = 50 in 2012 and n = 54 in 2015). Parasites were genotyped using 10 microsatellite markers. Results: Despite intensified control efforts, genetic diversity remained high (HE = 0.66–0.86) and was not significantly different among the four populations (P > 0.05). Specifically, HE slightly decreased from 0.76 in 2004 to 0.66 in 2016 at the CMB and increased from 0.80 in 2012 to 0.86 in 2015 at the TMB. The proportions of polyclonal infections varied significantly among the four populations (P < 0.05), and showed substantial decreases from 48.0% in 2004 to 23.7 at the CMB and from 40.0% in 2012 to 30.7% in 2015 at the TMB, with corresponding decreases in the multiplicity of infection. Consistent with the continuous decline of malaria incidence in the GMS over time, there were also increases in multilocus linkage disequilibrium, suggesting more fragmented and increasingly inbred parasite populations. There were considerable genetic differentiation and sub-division among the four tested populations. Temporal genetic differentiation was observed at each site (FST = 0.081 at the CMB and FST = 0.133 at the TMB). Various degrees of clustering were evident between the older parasite samples collected in 2004 at the CMB and the 2016 CMB and 2012 TMB populations, suggesting some of these parasites had shared ancestry. In contrast, the 2015 TMB population was genetically distinctive, which may reflect a process of population replacement. Whereas the effective population size (Ne) at the CMB showed a decrease from 4979 in 2004 to 3052 in 2016 with the infinite allele model, the Ne at the TMB experienced an increase from 6289 to 10,259. Conclusions: With enhanced control efforts on malaria, P. vivax at the TMB and CMB showed considerable spatial and temporal differentiation, but the presence of large P. vivax reservoirs still sustained genetic diversity and transmission. These findings provide new insights into P. vivax transmission dynamics and population structure in these border areas of the GMS. Coordinated and integrated control efforts on both sides of international borders are essential to reach the goal of regional malaria elimination. [ABSTRACT FROM AUTHOR]