This study reports on the socio-demographic and locality factors that influence ethnobiological knowledge in three communities of Zapotec indigenous people of the Isthmus of Tehuantepec, Mexico. It uses local botanical nomenclature as a proxy for general ethnobiological knowledge. In each of these communities (one urban and two rural), 100 adult men were interviewed aided with a field herbarium. Fifty had a background in farming, and 50 worked in the secondary or tertiary sector as their main economic activity, totaling 300 interviews. Using a field herbarium with samples of 30 common and rare wild regional species, we documented visual recognition, knowledge of the local life form, generic and specific names and uses (five knowledge levels measuring knowledge depth). The relationship between sociodemographic variables and knowledge was analyzed with simple correlations. Differences between the three communities and the five knowledge levels were then evaluated with a discriminant analysis. A general linear analysis identified factors and covariables that influenced the observed differences. Differences between the groups with different economic activities were estimated with a t-test for independent samples. Most of the relationships found between sociodemographic variables and plant knowledge were expected: age and rurality were positively related with knowledge and years of formal schooling was negatively related. However, the somewhat less rural site had more traditional knowledge due to local circumstances. The general linear model explained 70–77% of the variation, a high value. It showed that economic activity was by far the most important factor influencing knowledge, by a factor of five. The interaction of locality and economic activity followed. The discriminant analysis assigned interviewees correctly to their localities in 94% of the cases, strengthening the evidence for intracultural variation. Both sociodemographic and historic intracultural differences heavily influence local knowledge.