The objective of the study was to evaluate the effect of cold stress at flowering stage on plant structure, yield and chemical composition of seeds of 15 soybean cultivars. The study was conducted in 2019–2020, using the complete randomization method, in three replications. Fifteen soybean cultivars belonging to three maturity groups: early (EC), middle-early (MC) and late (LC) cultivars were included. Weekly cold stress (17/13 °C day/night) was applied at plant flowering stage. In the control treatment, plants were kept under natural conditions (24/17 °C day/night). Our research showed that cold stress negatively influenced the elements of plant structure: height, number of nodes, stem dry mass, number and weight of pods, number and weight of seeds per plant, as well as yield of soybean seeds, reducing it on average by 24%, as compared to the control treatment. The highest yield decrease was found in LC cultivars (31.2%), while a smaller and similar one in EC and MC cultivars (by 19.7 and 20.1%, respectively). Significant varietal differences were found for plant biometric traits and seed yield. EC cultivars had the lowest set first pod, as well as the lowest number of nodes, number of pods and seeds, pod and seed weight per plant, and seed yield. MC cultivars had the highest set first pod, and the smallest stem DM and seed yield average by 29.2% higher compared to EC cultivars. LC cultivars had the highest stem DM, number of pods and seeds, and pod and seed weight per plant compared to the other cultivar groups, and yield by 22.8% higher compared to EC cultivars. The experimental factors significantly affected crude protein, crude fat, and crude ash content, while they did not differentiate water-soluble carbohydrate and crude fiber content. Cold stress at the flowering stage caused a significant increase in protein content (by 4.1% on average) and ash content (by 3.8%) and a decrease in fat concentration (by 6.9%) in soybean seeds. Differences in nutrient content among cultivars were a genetic trait not related to cultivar maturity.