In this study, malebiased NUPTs (nuclear integrants of plastid DNA) were isolated and analyzed in the genome of Asparagus officinalis, a dioecious plant, by using genome substractive hybridization method. (1) 52 malebiased sequences with size ranged from 63 bp to 297 bp were obtained from the substractive hybridization library. Among these sequences, 19 were originated from chloroplast genome, which were designated as Ao1-Ao19. These sequences all showed high similarity (>84%) with the corresponding sequences in asparagus chloroplast genome, while Ao19 showed 100% similarity with the corresponding sequence in the asparagus chloroplast genome. (2) Genome semiquantitative PCR revealed that four (Ao1, Ao3, Ao10, and Ao18) out the 19 sequences were stable malebiased NUPTs. (3) Sequence alignment showed the NUPTs were mainly derived from the inverted repeat region (IR) (containing IRa and IRb) of the asparagus chloroplast genome, indicating that the sequences of IR region of chloroplast genome were more preferred to transfer to nuclear genome to form malebiased NUPTs sequences.