The potential introduction of the newly licensed HPV-vaccines at affordable prices could play a central role in preventing cervical cancer in middle and low-income countries, where most of the cases occur. In spite of this, basic epidemiological data about HPV and HPV-related lesions are still scarce. To determine the prevalence of cervical abnormalities, HPV prevalence, genotype distribution and related risk factors, we carried out a cross-sectional study in a population from Guatemala that included 297 women from the general population (GP women) and 297 sex workers (SW). All participating women were interviewed and underwent a complete gynecological examination that included collection of cervical cells for cytological diagnosis, HPV DNA detection and typing by PCR methods. Cervical HPV DNA prevalence among GP women and from SW was 38.1% (95% CI: 32.5-43.8) and 67.3% (95% CI: 61.7-72.6), respectively. The prevalence of abnormal cytology among GP women was 7.7% (ASCUS = 1.4%, LSIL = 4.7%, ASC-H = 0.3% and HSIL = 1.4%) and among SW was 21.6% (ASCUS = 7.5%, LSIL = 10.6%, ASC-H = 1.4% and HSIL = 2.1%). The most prevalent HPV types among women with normal cytology were HPVs 51 (n = 30), 66 (n = 25) and 16 (n = 25), and among women with HSIL or ASC-H (n = 14) HPVs 58 (n = 5) and 16 (n = 5). Determinants associated with HPV DNA detection were having had an occasional partner during the last 6 months and smoking habit among GP women, and being a minor among SW. HPV and abnormal cytology prevalence is high among women in Guatemala. The introduction of a HPV vaccination program would prevent an important fraction of HPV-related disease burden.