This study was carried out in the Tissue Culture Lab (TCL), in Al-Zahriya Garden at the Horticultural Institute of Agricultural Research Center in Giza, Egypt between 2018 to 2020. This study indicates the effect of different water sources and cytokinins during the proliferation stage of in vitro propagation Paulownia tomentosa plant. In this study, we do that by culturing tissues on epiphytic plants obtained from the same laboratory in the Zohriya garden. the first factor was the water source (tap and distilled water) Two different types of water sources were used: distilled water and tap water. Chemical analysis of tap water was carried out from pH TDS, TSS, EC NO3 and Ca, Na, and the second factor was the cytokinin treatments where three different types of cytokinins BA, Kin, and TDZ were used at different concentrations, 1mg/L, 2 mg/L 3mg/L 4mg/L and 5mg/L. At control concentration [0mg/L], The experiment was conducted by this study to find out the best protocol for in vitro micropropagation of Paulownia tomentosa. The results showed that the BA at 2mg/L got the highest rank for shoot length and shoot number and BA at 3mg/L got the highest rank of leaves number and shoot fresh weight while using Kin at 3mg/L got the highest rank for shoot length and total chlorophyll content. Therefore, we recommend using 2mg/L from BA with Kin at 3mg/L to get the highest rate of shoot length and shoot number and the highest rate of chlorophyll content.