Pathogenic fungi cause dying off of apple tree and giving negative impact to farmers. The objective of this study was to identify tree pathogenic fungi isolates from apple tree in the Batu City and to know phylogenetic relationship of the isolates based on the DNA sequences. The method in this study included subculture of three pathogenic fungi isolates (M1, M4, and MB1) using V8 Juice Agar medium for one to three days, continued with DNA isolation, amplification using ITS5/ITS4 primer, purification, sequencing, and sequence homology analysis of DNA amplicons of each isolates with reference isolates. The results showed similarity value of DNA sequence of the three isolates M1, M4, and MB1 of more than 99%. The three isolates M1, M4, and MB1 have a similarity value of DNA sequence with species of Pythium splendens of 99.84%, 99.67% and 99.83%, respectively. On the other hand similarity value between those isolates with Phytophthora was less than 76%. It was concluded that the three isolates are Py. splendens.