The objective of this study was to obtain a genetically stable haploid in vitro-derived line from Siberian larch (Larix sibirica Ledeb.) using megagametophyte explants, which then could be used for different molecular genetic studies, including whole genome de novo sequencing. However, cytogenetic analysis and genotyping of 11 microsatellite loci showed high levels of genomic instability and a high frequency of mutation in the obtained megagametophyte-derived callus cultures. All cultures contained new mutations in one or more microsatellite loci.