IntroductionAspergillussydowiiis an important filamentous fungus that inhabits diverse environments. However, investigations on the biology and genetics ofA. sydowiiin subseafloor sediments remain limited.MethodsHere, we performedde novosequencing and assembly of theA. sydowii29R-4-F02 genome, an isolate obtained from approximately 2.4 km deep, 20-million-year-old coal-bearing sediments beneath the seafloor by employing the Nanopore sequencing platform.Results and DiscussionThe generated genome was 37.19 Mb with GC content of 50.05%. The final assembly consisted of 11 contigs with N50of 4.6 Mb, encoding 12,488 putative genes. Notably, the subseafloor strain 29R-4-F02 showed a higher number of carbohydrate-active enzymes (CAZymes) and distinct genes related to vesicular fusion and autophagy compared to the terrestrial strain CBS593.65. Furthermore, 257 positively selected genes, including those involved in DNA repair and CAZymes were identified in subseafloor strain 29R-4-F02. These findings suggest thatA. sydowiipossesses a unique genetic repertoire enabling its survival in the extreme subseafloor environments over tens of millions of years.