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Soybean is planted in a wide span of the world, and flowering and maturity time is an important trait determining soybean yield formation and adaptation. Maturity loci <i>E1</i>, <i>E2</i>, <i>E3</i> and <i>E4</i> were frequently reported as the most influential genetic loci for soybean flowering and maturity. To understand the allelic variation and assess the phenological traits of cultivars with different <i>E</i> allelic combinations in natural environments, 251 cultivars of maturity group (MG) I–V were field tested in 42 locations across four sub-regions in the Huang-Huai-Hai and Northwest region of China and genotyped with KASP markers for <i>E1</i>–<i>E4</i> loci. The results indicated that mutant alleles were only found in the <i>E1</i> and <i>E2</i> locus, all of the cultivars carried functional alleles in the <i>E3</i> and <i>E4</i> loci in this area, with the frequency of mutant allele to be higher in early maturity groups (MGs) than late MGs. Among nine <i>E</i> allelic combinations in this area, one photoperiodic insensitive mutation in <i>E2</i> loci (<i>E1/e2-ns/E3-Ha/E4</i> and <i>E1/e2-ns/E3-Mi/E4</i>) made up the largest proportion (25.10 and 18.33%), while two photoperiodic insensitive mutations in both <i>E1</i> and <i>E2</i> loci (<i>e1-as/e2-ns/E3-Ha/E4</i>) (1.20%) occupied the lowest proportion in this panel. The major combinations of <i>E</i> locus for MGI, MGII and MG III in this area were <i>E1/E2-dl/E3-Mi/E4</i>, <i>E1/e2-ns/E3-Mi/E4</i> and <i>E1/e2-ns/E3-Ha/E4</i>, respectively. Cultivars carrying <i>e1-as/e2-ns/E3-Ha/E4</i> genotype flowered earliest (34 days) on average, 7.6 days earlier than the latest-flowering <i>E</i> haplotype (<i>E1/e2-ns/E3-Ha/E4</i>). This study provided an opportunity to detect the <i>E</i> allelic combinations in the Huang-Huai-Hai River Valley and the Northwest China, which would facilitate the improvement of soybean adaptation in the future.