Objectives: We present the evaluation of a methodology for the genotypic assessment of human immunodeficiency virus type-1 (HIV-1) drug resistance, optimized for use with dried blood spots (DBS). Methods: The ability to generate HIV-1 protease (PR) and reverse transcriptase (RT) contiguous amplicons and nucleotide sequences from DBS was evaluated. Different collection matrices and extraction methodologies were compared. The relative subtype sensitivity of the amplification strategy was assessed using a comprehensive panel of plasmids representing A-H subtypes. A panel of DBS and plasma specimens was subjected to HIV genotyping. Sequences generated from each sample type were compared. Results: Extensive replicate testing revealed most sensitivity with the use of 903 filter paper and silica/guanidine extraction, which had an estimated 95% inclusivity endpoint of 1542 proviral copies/mL, as compared with 21 573 proviral copies/mL for the FTA system. All HIV-1 group M subtypes analysed - with the exception of subtypes A2, AE, AG, F and H - had a relative sensitivity of ≤10 plasmid copies/PCR reaction. The PCR was multiplexed to include amplification of a human housekeeping gene to monitor the integrity of the human genomic DNA. Using a panel of clinical samples, we demonstrated the ability to amplify and sequence from 83% (n = 10) in the PR region and 100% (n = 12) in the RT region, of samples with detectable viral load. All specimens with an HIV-1 RNA load ≥1000 copies/mL were successfully amplified and sequenced. Twelve specimens had pol genotyping from both plasma and DBS samples. Sequence analysis and drug resistance interpretation revealed that 10 (83%) provided concordant drug resistance interpretation. Conclusions: Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy. © The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.