The DP‐305423‐1 transgene elevates oleic acid concentration and reduces polyunsaturated fatty acids in soybean [Glycine max (L.) Merr.] oil, resulting in increased oxidative stability. The objective of this research was to investigate the effect of the DP‐305423‐1 transgene on agronomic and seed traits using four backcross derived soybean populations to provide further understanding of the transgene's influence across varying genetic backgrounds and environments. An equal number of high‐oleic, low‐linolenic (HOLL) and normal‐oleic, low‐linolenic (NOLL) progeny from four BC1F4:F6 populations—B (n = 30), C (n = 22), D (n = 56), and E (n = 34)—were grown across six locations in 2015. Across all locations, the high‐oleic (HO) progeny had a mean yield 77 kg ha−1 less in Population B, and 382 kg ha−1 greater in Population C compared with the normal‐oleic (NO) class. There was no significant difference in yield between HO and NO in Populations D and E. The HO progeny had a significantly lower mean oil concentration and greater protein concentration than the NO class in all populations, whereas 100‐seed weight of the HO class was, on average, greater than in the NO class. The results provide evidence that the reduction in oil concentration is associated with the HO phenotype in the progeny carrying the DP‐3054231‐1 transgene and demonstrate no consistent effect of the transgene on seed yield, suggesting that differences may be due to the genetic background of the population studied.