Aim. CHI3L1 (chitinase 3-like protein 1) is a secretory glycoprotein highly upregulated in glioblastoma (GBM) and is recognized as a molecular marker of tumor mesenchymal subtype. However the gene’s short transcriptional isoform (CHI3L1Δ8) is poorly studied regarding its biological role in GBM. O⁶-methylguanine-DNA methyltransferase (MGMT) promoter methylation is considered a prognostic marker for temozolomide (TMZ) resistance in GBM patients, though some conflicting data exist. We aimed to analyze methylation of the MGMT promoter and CHI3L1/CHI3L1Δ8 expression in cells of the panel of U-251 MG (U251) glioblastoma cell lines, sensitive and resistant to TMZ. Conclusions. The obtained data indicate involvement of cellular mechanisms other than the reparative activity of MGMT in the evolution and maintenance of the TMZ-resistant phenotype. This is consistent with other studies on the decreasing of the prognostic role of the MGMT promoter methylation status for the assessment of therapeutic effect of TMZ in the treatment of glioblastoma. Balance alteration between the two CHI3L1 transcripts could be a part of the glioblastoma chemoresistance mechanism in response to TMZ treatment.