Identifying positions at which anticancer drug molecules incorporate into DNA is essential to define mechanisms underlying their activity, but current methodologies cannot yet achieve this. The thymidine fluorine substitution product trifluridine (FTD) is a DNA-damaging anticancer agent thought to incorporate into thymine positions in DNA. This mechanism, however, has not been directly confirmed. Here, we report a means to detect FTD in a single-stranded oligonucleotide using a method to distinguish single molecules by differences in electrical conductance. Entire sequences of 21-base single-stranded DNAs with and without incorporated drug were determined based on single-molecule conductances of the drug and four deoxynucleosides, the first direct observation of its kind. This methodology may foster rapid development of more effective anticancer drugs.