Background: This study aims to systematically evaluate the genetic divergence among 200 fresh maize inbred lines using both phenotypic and molecular markers, and to compare the efficacy of these two approaches for genetic classification. Methods: Phenotypic clustering analysis was conducted based on eight key agronomic traits, including plant height and ear length. Additionally, molecular analysis was performed using 40 Simple Sequence Repeat (SSR) primer pairs, resulting in the generation of 230 polymorphic alleles. The polymorphism information content (PIC) was calculated to evaluate the discriminatory power of the markers. Results: Phenotypic analysis categorized the inbred lines into four groups, comprising 25, 38, 97, and 40 lines, respectively, with benchmark lines distributed across Groups I and III. SSR analysis revealed a high level of genetic diversity, with an average of 5.75 alleles per locus and a mean polymorphic information content (PIC) of 0.70. Molecular grouping further divided the population into four distinct clusters, representing 26.5%, 51.0%, 14.0%, and 8.5% of the total, which exhibited different distribution patterns compared to the phenotypic grouping. The distribution of benchmark lines across various molecular groups confirmed their genetic divergence. Conclusions: SSR-based clustering demonstrated superior robustness and reliability compared to phenotypic grouping for genetic discrimination. These findings confirm the substantial genetic diversity present in fresh maize inbred lines and support the preferential use of SSR markers in maize breeding programs for precise genetic characterization.

This study measured eight key phenotypic traits across 259 fresh maize inbred lines, including plant height and spike length. A total of 82 single nucleotide polymorphisms (SNPs) significantly associated with these phenotypes were identified by applying a mixed linear model to calculate the best linear unbiased prediction (BLUP) values and integrating genome-wide genotypic data through genome-wide association analysis (GWAS). A further analysis of significant SNPs contributed to the identification of 63 candidate genes with functional annotations. Notably, 11 major candidate genes were identified from multi-trait association loci, all of which exhibited highly significant P-values (<0.0001) and explained between 7.21% and 12.78% of phenotypic variation. These 11 genes, located on chromosomes 1, 3, 4, 5, 6, and 9, were functionally involved in signaling, metabolic regulation, structural maintenance, and stress response, and are likely to play crucial roles in the growth and physiological processes of fresh maize inbred lines. The functional genes identified in this study have significant implications for the development of molecular markers, the optimization of breeding strategies, and the enhancement of quality in fresh maize.