Plant introductions (PIs) may increase the genetic variability of soybean populations and lead to greater genetic gains from selection. Five soybean populations containing different percentages of PI parentage were developed at Iowa State University to evaluate the use of PIs for increasing genetic variability for seed yield in a recurrent selection program. The objective of this study was to evaluate the performance and genetic variability of the Cycle 4 (C4) populations of AP 10 (100 % PI), AP 11 (75 % PI), AP12 (50 % PI), AP13 (25 % PI), and AP14 (0 % PI) for agronomic traits and seed composition. Multiplexing technology for simple sequence repeats (SSRs) was developed to facilitate the molecular diversity analysis of the elite and PI parents of AP 10 to AP 14. The mean performance of the C4 populations for seed yield was inversely proportional to the percentage of PI parentage that was initially present in CO. Genetic variance estimates for seed yield were similar among populations, indicating that the PIs did not provide greater genetic variability when recurrent selection was conducted for four cycles. AP 10 to AP 13 had higher means and more genetic variability for seed weight and, in particular, protein content, suggesting PIs may be valuable for the simultaneous improvement of yield and protein in a recurrent selection program. Eleven multiplex sets comprising 74 SSR markers were developed to analyze the molecular diversity of the PI and elite parents of AP 10 to AP 14. Results from the analysis showed greater diversity and less genetic similarity among the PIs than among the elite genotypes. The least amount of genetic similarity was between the PIs and elite genotypes. The ability of SSRs to distinguish among elite soybean genotypes and PIs with agronomic merit may facilitate the transfer of favorable alleles from PIs into elite populations.