The genetic relationships of Vietnamese black pepper varieties in the Central Highlands and Southern were evaluated by comparing the rbcL gene sequences of the collected black pepper varieties and the reference sequences on the base source NCBI data. Analysis results of nucleotide gene sequence variable rbcL of black pepper varieties showed Vietnamese varieties have much variable in nucleotide sequence compared to Asian varieties. The important characteristic variable areas of the Vietnam II black pepper group occurred at position 59292 (except for varieties SDL, VLDN5 and VLDN6), positions 59282 and 59295, variable appeared in 5 varieties (VLPQ1, VLVT1, VLGL3, VLDN4 and VLDN5), accounting for 41.67% of total samples collected. Genetic distances of black pepper varieties collected in the Central Highlands and the Southern, Vietnam (Vietnam II) (0.024 ± 0.005) with the Asian group were higher than those of black pepper group researched (Vietnam I) (0.001 ± 0.001). The phylogenetic tree demonstrated the difference between the two groups of Vietnamese black pepper. Among the varieties in the Vietnam II group, only the VLDN6 pepper variety has a close relationship with the Asian group. Analyzing the variable positions, the group Vietnam II was divided into 3 groups: Group I: VLPQ3; Group II: VLGL3, VLDN4 and VLDN5; Group III: SDL, VLVT1, VLVT2, VLDS1, VLGL1, VLPQ1 and VLPQ2. Thus, black pepper in the Central Highlands and Southern regions of Vietnam will be potential region for genetic diversity research in Asian pepper populations.