The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multi basic cleavage site (MBCS) in the HA of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space—including along the olfactory route—and the role of the MBCS in systemic replication. Intranasal inoculation with wild type H5N1 virus revealed extensive replication in the olfactory mucosa from where it spread to the olfactory bulb and the rest of the CNS, including the cerebral spinal fluid (CSF). Virus spread to the heart, liver, pancreas and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking a MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, the olfactory and hematogenous routes in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.