Cervical cancer, caused by oncogenic (high risk [hr]) human papillomavirus (HPV) subtypes, is the most common cancer in women in Guatemala and the most common cause of cancer mortality in women aged 15-44 years. Visual inspection with acetic acid (VIA) with onsite cryotherapy “test-and-treat” is recommended for underserved Guatemalan indigenous rural women. This research assessed: 1) hrHPV infection prevalence in women screened by VIA; 2) Sensitivity and specificity of VIA in detecting hrHPV infection and cytologically identified precancerous and cancerous lesions; and 3) Factors associated with hrHPV infection. Analysis of anonymous data collected during VIA clinics in 2013 (N = 205) and 2017 (N = 234) for indigenous women aged 21-65 years in six villages showed 22.6% (95% confidence interval [CI]=18.7%-27.2%) had hrHPV cervical infection. VIA results were abnormal in 5.9% (95%CI=3.8%-8.8%). Only nine VIA exams in 89 women with hrHPV were abnormal (Sensitivity=10.1%, 95%CI=4.7%-18.3%), although abnormal VIA was associated with hrHPV (Prevalence Ratio [PR])=1.8; 95%CI=1.1-3.1; P=.05). Of 221 women who had VIA, hrHPV nucleic acid testing and liquid preparation cytology (equivalent to Papanicolaou or “Pap”) testing, 10 (4.7% [95%CI=2.3%-8.5%]) had abnormal cytological results, including one cancer, four high- and five low-grade squamous intraepithelial lesions. VIA sensitivity and specificity for detection of precancerous cytological abnormalities and cancerous lesions were 20.0% (95%CI=2.5%-55.6%) and 96.0% (95%CI=92.3%-98.3%) respectively. In contrast, hrHPV sensitivity and specificity were 100% (95%CI=71.7%-100%) and 88.7% (95%CI: 83.9%-92.7%). In both years combined, women aged fewer than 29 years or reporting fewer than four pregnancies were more likely to have hrHPV cervical infection (36.8%, 27.3%, respectively) than those who were older or reported more pregnancies (18.7; P=.025, respectively); 60.0% reported some form of modern contraception. Progesterone injections or implant users were more likely to have hrHPV infection (31.9%) than women using other or no contraceptives (19.5%); PR=1.6; 95%CI=1.1-2.4; P=.01). These data suggest that VIA may not be sufficiently sensitive for use in cervical cancer screening. “Test-and-treat” screening using hrHPV real-time testing, as recommended by the World Health Organization may be preferable to VIA, and may be acceptable using self-collected specimens.