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Solanum nigrum is a noxious weed in agricultural ecosystem that limits many crops' production. The aim of the present study was to understand the level of genetic diversity and population structure of S. nigrum in China. A specific-locus amplified fragment (SLAF) sequencing method was conducted to detect single-nucleotide polymorphisms (SNPs) in the genomes of S. nigrum from 66 populations in China. A total of 616,533 high-quality SNPs were identified from 189,840 SLAFs, with an average sequencing depth of 10.59× fold and a Q30 value of 93% and a GC content of 42.78%. It showed a considerable amount of genetic diversity and genetic variability of S. nigrum among samples. The genetic differentiation of S. nigrum indicated that there was a low level of genetic differentiation (Fst < 0.1000) among geographical populations. A cluster analysis showed that populations of S. nigrum were divided into two subgroups, with some samples from adjacent position roughly clustered together, which showed some correlation between geographic origins. A population structure analysis suggested the 66 S. nigrum samples could have originated from three different genetic clusters. The Xinjiang site was the only location where all genetic clusters were found, which suggested these populations were genetically diverse. These results showed that there was a high degree of genetic diversity and low difference among the different geographical populations of S. nigrum. The results from the genetic structure of the SNP markers indicated that wide genetic variability exists among the population of S. nigrum in China, which may contribute to the adaptation and infestation of this weed species. [ABSTRACT FROM AUTHOR]