We identified the mitochondrial DNA haplogroups A, B, C and D in 75 present-day Maya individuals, 24 Maya individuals of the colonial period and one pre-Columbian Maya individual from Quintana Roo, Mexico. We examined these data together with those of 21 Maya populations accounting for 647 present-day Maya individuals and 104 ancient Maya individuals. A demographic study based on the analysis of fertility and endogamy was carried out in two modern Maya populations to identify cultural factors that influence the mitochondrial haplogroup genetic diversity. Most present-day and ancient Maya populations show a distribution pattern of mitochondrial haplogroup frequencies A, C, B and D in decreasing order, with haplogroup D absent in several populations. Considering only modern Maya populations with at least 50 individuals analyzed, the present-day Tzotzil and Lacandon populations from Chiapas show the higher and lower genetic diversities, 0.706 and 0.025 respectively. Our results show small genetic differences between the Maya populations with exception of the present-day Tojolabal and Lacandon populations from Chiapas. The present-day Lacandon population from Chiapas is different to other Maya populations in showing almost only haplogroup A. This result suggests a long history of isolation and endogamy as well as a possible founder effect inside the Lacandonian rain-forest. The contemporary Tojolabal population is the only one with an unusual mitochondrial haplogroup pattern exhibiting a frequency of haplogroup B higher than A and the absence of haplogroup C. With a small sample size, the pre-Columbian Copan Maya show a high content of haplogroup C and a low frequency of haplogroup D. The genetic homogeneity of the Maya populations is indicative of a common origin and nearly continuous gene flow in the longterm within a general isolation of the whole group, conversely to the Nahua populations that had different origins. Our demographic study showed high fertility rates and high levels of endogamy Pre-print version. Visit http://digitalcommons.wayne.edu/humbiol/ after publication to acquire the final version.Pre-Columbian Mayan society was characterized by a highly developed social structure and productive technology. The population size fluctuated geographically and temporally with a tendency to increase in the cities. The Maya were divided in autonomous political entities, maintaining multiple networks through commerce, warfare, and migration that influenced their economic, cultural, and demographic development (Renfrew 1975;Schele and Freidel 1990;Folan et al. 2000;Sierra et al. 2014).Evidence of far-reaching commerce, exchange of elite individuals through marriage, and non-elite population mobility suggests a degree of genetic flow among Mayan populations in the