Huanglongbing (HLB) is one of the most devastating diseases affecting citrus production worldwide. Although absent from Europe and Australia, the establishment of insect vectors in the Mediterranean basin represents a phytosanitary threat. While real-time PCR (qPCR) is considered the gold standard for HLB diagnosis, its implementation remains limited in many laboratories due to equipment and cost requirements. In addition, there is a lack of EPPO validated conventional PCR methods, species-level barcoding markers, and standardized reference materials for HLB diagnostics. This study aimed to validate two conventional PCR assays targeting the fusA and rpoB genes, evaluate their appropriateness as DNA-barcoding markers for HLB-associated 'Candidatus Liberibacter' species, and develop a safe biological reference material (RM), all in accordance with EPPO standards. The barcoding potential of both loci was evaluated using 664 sequences, and a plasmid-based RM was designed, characterized, and deposited in a public culture collection. Both PCR assays showed 100% inclusivity and exclusivity. Diagnostic sensitivity reached 80.8% for fusA and 73.9% for rpoB when compared with reference real-time quantitative PCR (qPCR) assays. The fusA PCR showed superior repeatability, reproducibility, and overall diagnostic performance, supporting its use as the preferred conventional PCR target for routine detection. Sequence analyses demonstrated clear species-level discrimination among HLB-associated species and other related taxa, supporting fusA as a dual-purpose marker for detection and identification, and rpoB as a complementary DNA-barcoding marker for species-level assignment. The biological RM was successfully validated as a positive amplification control for all evaluated EPPO-compatible PCR assays. To our knowledge, this study provides the first experimental evaluation of candidate DNA-barcoding loci for HLB-associated species and describes the first biological RM specifically developed to support HLB diagnostics, method validation, and quality assurance.