Abstract Background Stenotaphrum secundatum is an important grass with a rich variety of accessions and great potential for development as an economically valuable crop. However, little is known about the genetic diversity of S. secundatum, limiting its application and development as a crop. Here, to provide a theoretical basis for further conservation, utilization, and classification of S. secundatum germplasm resources, we used phenotypic and molecular markers (single-nucleotide polymorphisms, SNPs; sequence-related amplified polymorphism, SRAP; inter-simple sequence repeat, ISSR) to analyze the genetic diversity of 49 S. secundatum accessions. Results Based on seven types of phenotypic data, the 49 S. secundatum accessions could be divided into three classes with great variation. We identified 1,280,873 SNPs in the 49 accessions, among which 66.22% were transition SNPs and 33.78% were transversion SNPs. Among these, C/T was the most common (19.12%) and G/C the least common (3.68%). Using 28 SRAP primers, 267 polymorphic bands were detected from the 273 bands amplified. In addition, 27 ISSR markers generated 527 amplification bands, all of which were polymorphic. Both marker types revealed a high level of genetic diversity, with ISSR markers showing a higher percentage of polymorphic loci (100%) than SRAP markers (97.8%). The genetic diversity of the accessions based on SRAP markers (h = 0.47, I = 0.66) and ISSR markers (h = 0.45, I = 0.64) supports the notion that the S. secundatum accessions are highly diverse. S. secundatum could be divided into three classes based on the evaluated molecular markers. Conclusions Phenotypic and molecular marker analysis using SNP, SRAP, and ISSR markers revealed great genetic variation among S. secundatum accessions, which were consistently divided into three classes. Our findings provide a theoretical basis for the genetic diversity and classification of S. secundatum. Our results indicate that SNP, SRAP and ISSR markers are reliable and effective for analyzing genetic diversity in S. secundatum. The SNPs identified in this study could be used to distinguish S. secundatum accessions.
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