Persian lime (Citrus latifolia Tan.) is a crop of high economic and social relevance in Mexico due to its nutritional properties, associated health benefits, and particularly the foreign exchange generated through exportation. In recent years, the state of Veracruz has become the leading production region for this species at the national level. However, this crop is susceptible to several vascular diseases that cause significant economic losses in the citrus industry. Among these, Huanglongbing (HLB) is considered the most destructive disease, exerting the greatest impact on Persian lime production. This study involved the collection of Persian lime leaf tissues from different sources: field-grown plants, certified nursery plants, and plants previously sanitized through micrografting and thermotherapy processes. Genomic DNA was extracted from the collected samples. DNA integrity was verified by agarose gel electrophoresis and quantified using a NanoDrop One-C spectrophotometer. Quantitative Real-Time Polymerase PCR (qPCR) was performed using a QuantStudio One thermocycler and SYBR Green Master Mix as the fluorescent detection system. Samples were considered HLB-positive when the cycle threshold (Ct) value was below 35 during the quantification of Candidatus Liberibacter asiaticus. The results support the early, precise, and efficient detection of CLas by qPCR in Persian lime plants, thereby contributing to improved diagnostic and phytosanitary management strategies for this crop.