The diversity analysis of bacteria is useful for the environmental assessment of soil. Traditional molecular-based methods such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis achieve a low-resolution display of bacterial DNA fragments on a gel. To improve the resolution, a novel two-dimensional DNA gel electrophoresis (2-DGE) method was designed. This method can generate a high-resolution DNA map that facilitates the detailed analysis of soil bacteria. This map can be obtained by utilizing 2-DGE to separate genomic DNA fragments produced by polymerase chain reaction (PCR) amplification on the basis of chain length and G+C content. To develop this 2-DGE method further and to apply it to the assessment of bacterial diversity, we carried out a 2-DGE mapping of bacterial DNA fragments from different environmental soils and computed Shannon index as well as plotted rank-abundance curves on the basis of the relative intensity of each spot on the maps. DGGE mapping was also performed to compare the resolution of the two methods. 2-DGE mapping was capable of generating a higher resolution display by a factor of more than 2 using a DGGE fingerprint pattern on a piece of gel. Furthermore, the higher number of detected spots from the 2-DGE map enabled the assessment of differences in bacterial diversity in complex soil systems using a logarithmic normal rank-abundance plot.