The objective of this research was to study the relationship between the fatty acid content and the altitude of orchards and fruit maturity stage of avocados (Persea americana Mill. cv. Hass) at different locations in the department of Antioquia (Colombia). Orchards between 1,340 and 2,420 m a.s.l. were selected and the fatty acid profile and content of the fruits were analyzed. Oleic acid showed the highest percentage for all of the locations and its percentage decreased drastically at lower altitudes, meanwhile the percentage of palmitoleic and linoleic acids increased in these orchards. The oleic/palmitoleic, linoleic/palmitoleic, and oleic/linoleic indexes increased significantly at higher altitudes. Palmitoleic acid increased significantly with the maturity stage (dry matter content) of the avocados for all of the locations. Some fatty acids showed a high linear correlation with the fruit maturity stage, varying the type of acid or index with altitude. The fatty acid content is a variable to consider in future studies for a protected designation of origin (PDO), due to the close relationship observed with the geographical growing area and its importance to human health. More assessments must be taken over the course of years and harvests with more contrasting environments in order to obtain more robust information and feed the model.