Orchid gathering in the Southern Highlands of Tanzania became an income generating strategy for HIV/AIDS-affected households, particularly for children orphaned by HIV/AIDS. Being orphaned might have implications for children's local ecological knowledge leading to indiscriminate harvesting of both preferred and non-preferred orchids. Using qualitative and quantitative mixed methods, we assessed the knowledge of wild orchid species among gatherers in four categories: children orphaned by HIV/AIDS, children not affected by HIV/AIDS, HIV/AIDS-affected adults, and non-HIV/AIDS-affected adults. Data collection took place from February 2006 until February 2007. More than 70% of the children orphaned by HIV/AIDS relied on tuber taste and texture to distinguish preferred and non-preferred species. HIV/AIDS-affected adults were competent at using orchid morphology to distinguish preferred from non-preferred orchid species. Gatherers stated preferred orchid species were primarily found in restricted areas, and non-preferred species were widespread. Cramer's V analysis revealed an association between the HIV/AIDS status of a gatherer and the gatherer's views on the conservation status of preferred and non-preferred species. Gatherers perceived non-preferred species as having higher natural regeneration potential than preferred species. Cramer's V analysis also showed a difference in gathering knowledge acquisition between HIV/AIDS-affected double orphans who emigrated from urban settings, and those residing in their natal rural village under guardian care. Both types of orphans relied primarily on other children for gathering knowledge, but those with guardians also gleaned knowledge from guardians. Across categories, children also gained knowledge from middlemen, who buy and resell the preferred orchids.