Serious pine needle disease, Dothistroma needle blight (DNB), caused by Dothistroma septosporum and D. pini was detected in Ukraine in 2004-2005, although D. septosporum was first reported in Ukraine in 1914. The aim of this study was to identify the Dothistroma species present on new hosts in Ukraine using different molecular techniques to increase our understanding of the local distribution of these pathogens. The current situation of Dothistroma needle blight in Ukraine was studied in native, planted pine forests and arboretums. The occurrence and distribution of DNB were studied in 2012–2018 and 480 needle samples were collected from 16 different regions in 96 localities, and the presence of DNB was confirmed in 62 of them. The host range of DNB consisted of 8 pine species including 3 subspecies and 2 spruce species, among them Pinus nigra J.F. Arnold subsp. pallasiana (Lamb.) Holmboe and P.sylvestris L. were the most frequent hosts. Results showed that both D. septosporum and D. pini were present on P. nigra subsp. pallasiana on the same trees and even in the same needles Moreover, D. septosporum was found first in Ukraine on Pinus ponderosa Douglas, Pinus banksiana Lamb and Pinus contorta Douglas in the arboretum as well as Picea pungens Engelm and Picea abies (L.) H. Karst. The suitability for the disease in the different forest types and intensity of the disease are discussed. To sum up, DNB was found in many parts of the country although the disease intensity is low, probably due to the unfavourable climate conditions for DNB development.