The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN2) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN2 related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA®) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method. A protocol for extraction of BoHV-1 DNA from FTA® card spotted with extended semen was optimized. The viral DNA was found to be stable on FTA® card for at least 28 days when the cards are stored at 4°-37 °C. The analytical sensitivity for the assay was determined using variable dilutions of BoHV-1 spiked semen and positive plasmid harbouring gB gene (97bp) spotted onto FTA® card and it was found to be 100.8 TCID50/ml or 100 copies respectively in real-time PCR. The test could detect as low as 100.008 TCID50/ml or 1 copy of positive plasmid when more number of replicates (n = 6) of the same sample were tested. This sensitivity was found to be comparable to Chelex method and both the methods demonstrated a very strong correlation (r = 0.9774; 95% CI: 0.9620-0.9860) in terms of Ct value (p < 0.0001). The diagnostic sensitivity and specificity of the FTA method in comparison to the Chelex method was 83.08% (95% CI: 71.73%-91.24%) and 93.23% (95% CI: 89.38%-96.01%) respectively when 316 samples were screened by both the methods. The degree of agreement between these two tests was good (Kappa value: 0.738; 95% CI: 0.646-0.829). The method was found to be robust and highly repeatable in inter-assay and intra-assay precision testing. The result suggests that the FTA® card holds promise as an alternative system for transportation of EFS for downstream screening of BoHV-1 DNA.