Benthic diatoms are ubiquitous in tidal flats and play major roles in maintaining coastal ecosystems. Spatio-temporal variations in diatom diversity have not been well-studied, mainly because of difficulties in morphological identification and the lack of appropriate genetic tools. To overcome these problems, we used the gene encoding the ribulose bisphosphate carboxylase large-subunit (rbcL) as a molecular marker, and sequenced these genes with the aid of the MiSeq platform. In this manner, we explored the genetic diversity of benthic diatoms in tidal flats of Guenso Bay on the west coast of Korea; differences in the spatial distributions of benthic diatoms were evident. The diatom communities were dominated by Nitzschia, Navicula, and Amphora; their relative distributions were affected by the sand proportion, grain size, and air exposure time. Our results suggest that meta-barcoding of the rbcL gene and next-generation sequencing can be used to explore the diversity of benthic diatoms.