Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL‑V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL‑V8B exhibits increased sensitivity to various DNA‑damaging agents, including compounds leading to DSBs formation (bleomycin and 6‑thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400‑fold), which is typical for HR‑defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL‑V8B cells. However, in contrast to other known HR mutants, CL‑V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL‑V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC‑induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL‑V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL‑V8B to crosslinking agents results in S‑phase arrest (as in the wild type cells), but also in larger proportion of G2/M‑phase cells and apoptotic cells. CL‑V8B exhibits similarities to HR‑ and/or FA‑defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.