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Background HIV early invades the central nervous system during primary infection, and compartmentalized viral replication in this district may lead to divergent viral evolution. HIV strains displaying a particular tropism in cerebrospinal fluid (CSF) have been highlighted in the past, mainly associated with amino acid (AA) patterns in the V3 loop of the gp120 encoded by the env gene. The aim of the study is to compare near full-length (NFL) sequences from CSF and plasma (PL) of the same subject in order to find possible new determinants of neurotropism.Materials and Methods Fifteen paired CSF PL samples from ART naïve chronically infected PWH were analysed. Ion Torrent NGS NFL sequencing was performed; reads were mapped to HXB2, and CSF PL consensus sequences were generated. Phylogeny was used for subtype assignment. AA alignments were obtained for gag, pol and env. CSF PL differences were categorized per position and compartments were compared using non parametric paired tests. Recurrent CSF-PL discordant sites (hotspots) were screened at position level. Positions associated with p24 region, pol transmitted drug resistance (TDR), V3-tropism, and fostemsavir (FTR) related gp120 substitutions were also analysed.Results Individuals were mainly male (86.6%), with a median (IQR) age of 52 (46-63), 60.0% were Italian. Median (IQR) log10 HIV 1 RNA copies/mL were 5.21 (4.63–5.89) in CSF and 4.79 (4.24–5.57) in PL. Subtype B prevalence was 66.7%. NFL was obtained in all paired samples (approximately 1- 9,719 nt), with at least 93.3% position covered, and with a mean number of mapped reads of about 1 million. AA alignments of CSF-PL NFL consensus highlighted a higher diversity in env compared with pol and gag. Recurrent CSF-PL hotspot residues in gp120 at 175/337/343 (n = 7 pairs each), and at gp120 positions 335/336/363/465 and gp41 positions 580/644/835 (n = 6) (p<0.05) were identified. By contrast, pol exhibited only a few discrete peaks, restricted to RT positions 306/433 (n = 2 and 3) and RNaseH 590/653 (n = 2 each) (p<0.05), consistent with low-density hotspot profile. For gag, no position reached significance, including in p24 region. Regarding V3 region, approximately half of the pairs showed an X4 tropism, and no evidence of compartmentalization was observed: X4 was discordant in only one pair (CSF only). Similarly, FTR-related substitutions (S375N/T, M426L, M434I) were largely concordant between compartments. TDR to NRTI (T69D, L74V) was rare and showed no significant CSF-PL differences. Moreover, no significant sites showed a compartment-specific pattern.Conclusions Despite the presence of localized env hotspots, CSF-PL discordances were limited and did not indicate a consistent compartment-specific divergence. Across env, pol and gag, along with V3 tropism markers, FTR-related sites and pol TDR, CSF-PL sequences remained largely concordant, suggesting overall genetic continuity between compartments rather than true divergent evolution.Figure 1Abstract P240 Figure 1Hotspot analysis of amino-acid differences between CSF–plasma paired samples across gag, pol, and env