To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.