The effect of the geographical origin on the polyphenolic composition of peppermint was investigated. Three independent biological replicates were used for Erbil and Tikrit respectively, and the pre-flower leaves of Mentha × piperita L. were analyzed using High-Performance Liquid Chromatography coupled with Diode-Array Detection (HPLC–DAD). Eleven major compounds were detected, such as Eriocitrin, Luteolin-7-O-rutinoside, Luteolin-7-O-β-glucuronoside, Narirutin, Isorhoifolin, Diosmin, Hesperidin, Lithospermic acid, Rosmarinic acid, Caffeic acid and Eriodictyol. The overall levels of the identified compounds were significantly higher in Erbil than in Tikrit, with a value of 491.31 ± 3.50 mg/g dry extract compared to 455.88 ± 3.41 mg/g dry extract, which represents a 7.21% decrease. Eriocitrin was the most abundant compound in both regions with 291.31 ± 3.12 mg/g in Erbil and 266.83 ± 3.08 mg/g in Tikrit. The second one was rosmarinic acid with 58.31 ± 0.87 and 51.87 ± 0.76 mg/g respectively. The levels of six compounds were significantly lower in the Tikrit samples compared with the Erbil samples, and four of the compounds were significantly higher in the Tikrit samples than in the Erbil samples; Eriodictyol was not significantly different. The largest increase was seen for Lithospermic acid (+20.48%) while the greatest decrease was observed for Hesperidin (-13.32%). The same compounds were found in both areas, but the amount of each compound was much higher in one area than the other. These findings show that geographical and environmental factors have a significant influence on the polyphenolic profile of peppermint and the importance of the use of multiple chemical markers for quality assessment and extract standardization.