Purpose We aimed to evaluate HIV-1 compartmentalization between the cerebrospinal fluid (CSF) and plasma and investigate as to which extent HIV-1 strains in CSF differ from those in blood and whether a correlation with either plasma viral load (pVL) or an altered blood–brain barrier (BBB) does exist. Study design We retrospectively evaluated paired CSF/blood samples collected from 86 HIV+ patients. HIV-RNA quantification, pol (PR/RT), and V3 sequencing were performed. HIV coreceptor tropism (CRT) was inferred (g2p, false-positive rate 10%, FPR). Data of standard CSF analysis were also reviewed; an altered CSF/plasma albumin ratio signified BBB damage. Neurological abnormalities (NA) were recorded. Results Overall, 32% of patients had a CSF/plasma HIV-RNA ratio > 1 ( discordance ); 3% of patients had detectable CSF HIV-RNA despite suppressed pVL ( escape ). Discordance was more frequent in ART-treated patients ( p  < 0.001) and in patients with NA ( p  = 0.016), but was independent of BBB damage ( p  = 0.65) and AIDS diagnosis ( p  = 0.96). Finally, CSF/plasma discordance was significantly more frequent ( p  < 0.0001) in patients with lower pVL values (< 10.000 copies/ml). Env divergence > 10% was found in 44% of sequences and was associated with ART ( p  = 0.008) and NA ( p  = 0.037). Overall, 24% of patients had a discordant CSF/blood CRT. A 100% nucleotide identity was observed in only 7.3% of pol sequences; notably, 10% of patients had resistance-associated mutations in CSF, but not in blood. Conclusions Our data confirm an independent replication and evolution of HIV within the CSF. A number of factors either hinder or contribute to the compartmentalization of HIV.