Days to flowering and maturity are controlled by genes E1-E7 and J in soybean. Previous studies revealed that E1-E5 and E7 influence tolerances to low-temperature-induced seed coat browning in different directions at various intensities. The E4 locus is useful for the development of early maturing cultivars with chilling tolerance because the recessive allele conditions both the early-maturing habit and chilling tolerance. This study was conducted to obtain a fine map of E4 by amplified fragment length polymorphism (AFLP) analysis using a F(8:9) family segregating for E4 that was developed from a cross between photoperiod-insensitive Japanese landraces, Sakamotowase (E4) and Miharudaizu (e4). AFLP analysis using a total of 4096 primer pairs detected 20 polymorphic markers between near-isogenic lines for E4. Linkage mapping incorporated 16 AFLP markers into a previously constructed genetic map around E4 in linkage group I. Eight AFLP markers were localized to unfilled areas between E4 and the closest markers identified previously. Two AFLP markers flanking E4, e48m41-8 and e18m38-8, were mapped at positions 0.6 and 5.4 cM apart from E4, respectively. They were dominant and in cis arrangement with the recessive allele (e4) conditioning the photoperiod insensitivity and chilling tolerance. These markers can be used in developing more precise markers for fine mapping and marker-assisted selection and in isolating the underlying gene via genome walking approaches.