Naturally occurring amino-acid substitutions in the hepatitis B virus (HBV) polymerase gene may be responsible for resistance to nucleoside/nucleotide (NUCs) analogues. To date, only pre-existing lamivudine resistance has been extensively studied. The aim of the present study was to determine the naturally occurring or pre-existing amino-acid substitutions related to NUCs in treatment naive Turkish patients with chronic hepatitis B (CHB). The investigation involved a total of 88 patients (65 males and 23 females; mean age, 34 years; range, 15-61 years) who were diagnosed with CHB between April 2008 and January 2009. According to HBeAg status, 66 patients were HBeAg-negative and 22 patients were HBeAg positive. Naturally occurring substitutions in the HBV polymerase region were detected by DNA sequencing in 17 (19%) and 30 (34%) patients, based on manual and geno2pheno tool database interpretation, respectively. Each amino-acid substitution appeared alone and included rtA194T, rtV214A, rtQ215S, rtI233V and rtN236T. The median values for viral load, ALT and AST were 3.3 log(10) (2.0-6.0) IU/mL, 36 (12-515) U/L and 27 (13-284) U/L, respectively, but these did not correlate with the observed amino-acid substitutions in the polymerase region. By direct sequencing, genotype D of HBV was found to still be dominant among Turkish patients. In conclusion, every patient who is diagnosed with CHB should be monitored before the start of treatment for more effective management of patient treatment options.