Background: Beyond bacteria, the human gastrointestinal tract is host to a vast diversity of fungi collectively known as the gut mycobiome. Little is known of the impact of geography, ethnicity, and urbanization on the gut mycobiome at a large population level. This is the first study that aims to delineate the configuration of human gut mycobiome across geography, ethnicity, and urbanization, in a large population. Method: We enrolled 942 Chinese subjects from different regions (Hong Kong and Yunnan) and 6 ethnic groups in Yunnan, including Han, Zang, Bai, Hani, Dai, and Miao, for each of which both rural and urban residents were included. The demographics data and metadata for study subjects were collected. Hong Kong is a densely populated urban city where all recruited subjects were exclusively Chinese Han. Yunnan is a multi-Chinese ethnicity cohabiting province with an exquisite population distribution feature, where urban subjects for all ethnicities co-reside in the provincial capital city Kunming and each of their originated ethnic group is distributed in a confined rural region. We enriched fungal DNA from the feces and performed ultra-deep metagenomics sequencing. Both the mycobiome and bacterial microbiome were profiled and compared between regions, ethnicities and rural versus urban residency. Correlations between gut fungi and bacteria were also assessed. Results: We found that geography was the strongest factor in shaping the gut mycobiome (PERMANOVA test, p<0.001). Hong Kong population had a marked depletion of fungal species (38 for Hong Kong vs 55 for Yunnan, mean fungal richness index), particularly symbiotrophic fungi, and a higher abundance of saprotrophic and pathotrophic fungi, compared to all populations in Yunnan. Gut mycobiome in Yunnan varied substantially as a function of ethnicity and rural/urban residency. Han and Zang ethnicities exhibited enrichment of various fungal species whereas Miao ethnicity had the lowest mycobiome diversity among all sampled populations. Overall, the genus Saccharomyces was enriched in urban populations in contrast to that a rich array of fungal genera were enriched in rural populations. Trans-kingdom correlation analysis between gut fungal richness and bacterial richness identified a positive correlation (Pearson correlation Rho= 0.985, p<-2.2e-16). The fungal species Densospora sp was the most correlated fungus with a large number of both positive and inverse correlations with gut bacteria and fungi. Conclusion: Our data for the first time highlight that geography, ethnicity and urbanization all impact the human gut mycobiome composition and contribute to population-level heterogeneity with decreasing effect size. The relationship between different gut mycobiome configurations and human health consequences warrants further investigation.