We investigated the degree of cross-contamination during the punching of FTA cards. Dried or wet FTA cards containing buccal cells were punched with a “Harris Micro Punch”. New FTA micro cards were then punched by the puncher that had either been cleaned by punching clean blotting papers or that had not been cleaned. To detect cross-contamination, DNA was extracted from the new FTA micro cards and was quantified by a Real-Time PCR assay with a detection limit of 0.1 pg/μl. DNA was also amplified using the Identifiler kit to perform short tandem repeat (STR) typing.   In the Real-Time PCR assay, for the dried FTA cards, DNA was detected in 2 and 3 samples out of 20 samples from the cleaned and the non-cleaned puncher, respectively. For the wet FTA cards, DNA was detected in 3 and 17 samples out of 20 samples from the cleaned and the non-cleaned puncher, respectively. The non-cleaned puncher, after punching the wet FTA card, gave the maximum DNA concentration (0.4 pg/μl). In the STR typing, 2 of 20 samples showed peaks up to approximately 60 RFU (relative fluorescent units) for the non-cleaned puncher after punching wet FTA cards. However, samples produced under the other three conditions showed no peaks above 50 RFU.   In conclusion, it was thought that the puncher did not need to be cleaned for the STR typing, when FTA cards were dried. In addition, when FTA cards were wet, the cross contamination up to approximately 60 RFU was observed in the STR typing without the cleaning of the puncher, but was reduced by the cleaning.