Medicago polymorpha is a successful invasive plant and widespread around the world with its ability to adapt to the novel environment conditions quickly. However, the evolutionary dynamics after invasion still largely unclear so far. In this study, we performed a specific-length amplified fragments sequencing (SLAF-seq) of ten representative Medicago polymorpha accessions enrolled from different regions in China to infer the population structure and nucleotide diversity. As a result, total 52,237 high-quality cross-species SNPs were identified. Population structure analysis based on these SNPs suggested entire Medicago polymorpha accessions could be divided into three groups, that is consistent with wild, intermediate, and cultivated. Moreover, a low level of gene flow occurred between cultivated and wild subgroups, and the wild subgroup had a higher allelic variation than the cultivated subgroup, which indicated that a genetic bottleneck may occurred during Medicago polymorpha domestication. The genotypic structure is no significant correlation with the geographic parameters (longitude and latitude) by Mantel test, suggested a rapid evolution to adapt to different environments. Additionally, selective sweep analyses found some improvement-selective genes. Gene function analysis revealed that they were primary involved in endonuclease activity and RNA-DNA hybrid ribonuclease activity biological processes, which are correlated with plant evolution. Therefore, we speculated these genes identified from selective sweep regions may have contributed to Medicago polymorpha domestication from wild to cultivated. Taken together, our results suggest that Medicago polymorpha has rapidly evolved across the invaded range. This will help us better understand the domestication history and intraspecific relationship of Medicago polymorpha, and provide basic information for breeding research in the future.

Table S1: Quality assessment of sequencing data.

Table S2: Number and coverage of SLAFs for each accession.

Table S3: Discovery results of SLAF markers.

Table S4: Evaluation of SNP for each accession.

Table S5: Distribution of minor allele counts.

Table S6: Means for six morphological traits recorded for ten Medicago polymorpha accessions.

Table S7: SLAF tags selected by purifying selection from cultivated to wild population.

Table S8: Plant accessions used in this study. Number and coverage of SLAFs for each accession.

Figure S1: Distribution of minor allele counts. The x-axis represents the minor allele frequency (MAF), y-axis represents the minor allele counts.

Figure S2: Rooted phylogenetic trees of Medicago polymorpha accessions.