With the characteristics of insect-borne transmission and systemic infection, Paulownia witches’-broom (PaWB) disease has been considered a serious epidemic disease in Taiwan. PaWB phytoplasma (PaWBP) seriously weakens paulownia (Paulownia taiwaniana) plants, and has an enormous influence on paulownia cultivation. To accurately diagnose PaWB, a rapid and reliable detection method, a polymerase chain reaction (PCR)-based analysis using primers derived from PaWBP 16S ribosomal (r)DNA sequences, was previously established. We used the PCR assay to trace the incubation periods of PaWBP, symptom expression stages of infected paulownia, distribution and concentration of PaWBP in paulownia trees, and insect vectors. The incubation period of PaWBP-infected paulownia (10 cm in height) was about 3 mo; distributions of PaWBP in the mesophyll, petioles, veins, and bark of paulownia plants were from high to low, respectively according to the PCR assay. We also found that it took about 3 mo for PaWBP to move from the top grafting site to the basal root in 30-cm-tall paulownia plants. In this study, it was proven that Halyomorpha mista could successfully transmit PaWBP from diseased paulownia to healthy paulownia seedlings, and detection of PaWBP in H. mista was confirmed by the PCR as well. Field tracking showed 2 population peaks of H. mista during the year: March to April and August to October. These results may be helpful in insect vector control in the future. In resistance experiments, paulownia hybrids also showed higher resistance than purebred plants.