There has been considerable interest in composition of gut microbiota in recent years, leading to a better understanding of the role the gut microbiota plays in health and disease. Most studies have been limited in their geographical and socioeconomic diversity to high-income settings, and have been conducted using small sample sizes. To date, few analyses have been conducted in low-income settings, where a better understanding of the gut microbiome could lead to the greatest return in terms of health benefits. Here, we have used quantitative real-time polymerase chain reaction targeting dominant and sub-dominant groups of microorganisms associated with human gut microbiome in 115 people living a subsistence lifestyle in rural areas of Papua New Guinea. Quantification of Clostridium coccoides group, C. leptum subgroup, C. perfringens, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus, and Lactobacillus spp. was conducted. Principle coordinates analysis (PCoA) revealed two dimensions with Prevotella, clostridia, Atopobium, Enterobacteriaceae, Enterococcus and Staphylococcus grouping in one dimension, while B. fragilis, Bifidobacterium and Lactobacillus grouping in the second dimension. Highland people had higher numbers of most groups of bacteria detected, and this is likely a key factor for the differences revealed by PCoA between highland and lowland study participants. Age and sex were not major determinants in microbial population composition. The study demonstrates a gut microbial composition with some similarities to those observed in other low-income settings where traditional diets are consumed, which have previously been suggested to favor energy extraction from a carbohydrate rich diet.