This work assessed the sanitary status of 46 Apulian Malus domestica varieties to preserve healthy Apulian germplasm sources and identify potential candidate clones that could be used in future certification program of autochthonous varieties in Puglia. The research was conducted as part of the Regional Project “Re.Ge.Fru.P”. The EU commission Directive 2020/217 provided the basis for this study by setting out a list of viruses and viroids necessary for the certification of Malus domestica. The analysis focused on detecting the presence of six specific viruses (ASGV, ACLSV, ASPV, ARWV1, ARWV2, ApMV) and two viroids (ASSVd, ADFVd), as well as a quarantine virus (ToRSV) and an EU-regulated virus (CLRV) known for their damages on apple trees. Furthermore, ten economically important viruses and viroids (MdoVA, CCGaV, ApNMV, PNRSV, ALSV, AFCVd, CRLV, AGCaV, ALutV, AHHVd), not included in the certification program, were screened. PCR analysis revealed positive results for AGCaV and ASPV in 61% of the samples, ACLSV in 57% of the samples, ARWV2 in 15% of the samples, ASGV in 12% of the samples and CCGaV, ALutV, and ARWV1 in 2% of the samples. This study presented compelling evidence of the first detection of ALutV and the first identification of AGCaV on apples in Italy. HTS reconfirmed the presence of AGCaV, ASPV, CCGaV, ARWV1, ARWV2, and ASGV with nucleotide identities ranging from 80.2% to 100%. A hitherto novel virus similar to Cherry virus Trakiya (CVT) was discovered in HTS and was confirmed with PCR using specific primers, with 46% nucleotide identity to CVT. These findings provide important insights into the sanitary status of the Apulian Malus domestica collection and lay the foundation for future certification programs and preservation efforts in Puglia.