Viruses and viroids can cause economic losses in fruit trees by reducing growth, vigor and yield. Efficient and reliable diagnostic methods are critical in identifying viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available PCR-based assays for pome and Prunus viruses and viroids (16 different pathogens) and developed new reverse transcription quantitative PCR (RT-qPCR) assays when current tests were inadequate. Thus, published PCR assays for targeted pathogens were evaluated in silico, using GenBank nucleotide sequence data, to determine their ability to detect specific virus and viroid isolates. Assays with nucleotide mismatches in the primer sequences were selected for modification. Modifications consisted of either the addition of primers/probes to the existing assay or a new assay design targeting a more conserved region of the virus/viroid genome. To validate proposed assay designs, select fruit tree populations were screened for targeted viruses and viroids via high throughput sequencing (HTS) compiling a representative set of isolates. Consequently, all the updated or new RT-qPCR assays successfully detected the different pathogens, including the diverse isolates. The development of robust, sensitive and reliable detection methods is needed for large scale pathogen testing, which will contribute to maintain the highest quality nursery stock.