The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almería, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome. © 2014 Springer Science+Business Media Dordrecht.