Even though calanoid copepods produce diapausing eggs that stay alive in lake sediments, these eggs have been rarely used in paleoecology since they lack diagnostic features. Thus we tried to develop a method to identify copepod diapausing eggs in Japan for helping in reconstruction of past lake food-web more precisely. First, a 28S rDNA (i.e. nc28S) barcode (ca. 300bp) was developed with using ethanol-fixed calanoid copepods collected from whole parts of Japan. Second, it was found that DNA could be extracted from an individual diapausing egg by the HotSHOT plus Sonic method. Finally, the nc28S region of diapausing eggs collected from various lakes was sequenced and compared with the nc28S barcode for species identification. We found that despite the short length the nc28S region could completely delimit Japanese freshwater calanoid copepods, suggesting that the region does work as the DNA barcode. All of the recovered nc28S sequences from diapausing eggs matched a certain copepod species involved in the barcode set. Thus we could develop a set of protocols (i.e. preparing a complete DNA barcode and sequencing egg DNA) that enables us to use copepod diapausing eggs in paleoecological studies in Japan. The nc28S barcodes may have a strong potential in paleoecology to uncover the paleodiversity of copepod not only in Japan but also in other parts of the world.