Lecanosticta acicola is an ascomycete that causes brown spot needle blight of pine species in many regions of the world. This pathogen is responsible for a major disease of Pinus palustris in the USA and is a quarantine organism in Europe. In order to study the genetic diversity and patterns of spread of L. acicola, eleven microsatellite markers and two mating type markers were developed. An enrichment protocol was used to isolate microsatellite-rich DNA regions and 18 primer pairs were designed to flank these regions, of which eleven were polymorphic. A total of 93 alleles were obtained across all loci from forty isolates of L. acicola from the USA with an allelic diversity range of 0.095 to 0.931 per locus. Cross-species amplification with some of the markers was obtained with L. gloeospora, L. guatemalensis and Dothistroma septosporum, but not with D. pini. Mating type (MAT) markers amplifying both idiomorphs were also developed to determine mating type distribution in populations. These markers were designed based on alignments of both idiomorphs of nine closely related plant pathogens and a protocol for multiplex PCR amplification of the MAT loci was optimised. The MAT markers are not species specific and also amplify the MAT loci in Dothistroma septosporum, D. pini, L. gloeospora and L. guatemalensis. Both types of genetic markers developed in this study will be valuable for future investigations of the population structure, genetic diversity and invasion history of L. acicola on a global scale. ; http://link.springer.com/journal/13313 ; Financial support to Josef Janoušek from the AKTION Czech Republic – Austria (project 58p23), the Scholarship Foundation of the Republic of Austria (OeAD-GmbH, Austria),theHlavka Foundation (Czech Republic; for internship at Massey University, New Zealand) and the Intern Grant Agency of the Faculty of Forestry and Wood Technology (Mendel University in Brno, Czech Republic).The project was supported financially by COST CZ LD12031 (DIAROD), the FPS COST Action FP1102 (DIAROD) and the European Union’s Seventh Framework Programme FP7 2007–2013 (KBBE 2009–3) under grant agreement 245268 ISEFOR.