Abstract 1. Avian sexing by polymerase chain reaction (PCR) plays an important role in sexual identification of avian species with similar phenotypes. Dried blood spots (DBSs) on paper blotters can help reduce the cost and problem of sample transportation and processing. 2. In the first experiment, several kinds of papers were evaluated for collecting DBS for chicken sexing by direct PCR with different processing methods. The most practical method with cost optimality was the utilisation of Whatman grade 1 filter paper with the combination of methanol fixation and boiling. 3. A second experiment was performed to determine whether cross-contamination could occur among samples cut with the same scissors. No-clean scissors were compared with ones cleaned with bleach-ethanol combination or 0.3N HCl. The PCR results showed that all three methods provided correct amplicon sizes without any false-positives regardless of the utilisation of cleaning intervention. 4. In conclusion, a technique that is suitable for DBS collection for avian sexing by direct PCR with cost efficacy was developed, and it was also shown that the utilisation of the same pair of scissors for several DBS samples did not affect the PCR results.