Background Generic substitution is a good approach to reduce pharmaceutical expenses without compromising healthcare quality. Yet, the practice of generic substitution has been contentious due to concerns on quality and efficacy. Objective This study was aimed to assess knowledge, attitude and practice among pharmacy professionals toward generic substitution in community drug retail outlets in Jimma town, Southwest Ethiopia. Methods A descriptive cross-sectional study was conducted among pharmacy professionals working in community drug retail outlets. Data was collected using a self-administered questionnaire. Knowledge was tested using a 3-point response format consisting of “Yes,” “No” and “I am not sure.” Attitude was evaluated using the 5-point Likert scale ranging from 1(strong agreement) to 5 (strong disagreement). The practice was examined as never, seldom, sometimes, often, and always with scores ranging from 0 to 4. The influences of socio-demographic factors on knowledge, attitude, and practice were tested using the Mann–Whitney U and Kruskal–Wallis tests as appropriate. P ≤ 0.05 was considered statistically significant. Results The mean knowledge score of participants regarding generic medicines was 5.75 ± 1.79. Only 32 respondents (30.2%) of the participants were knowledgeable about the generic substitution. 54 (50.9%) of respondents had positive attitude toward generic substitution and 52 (49.1%) had practiced generic substitution. The year of experience had a significant effect on knowledge (X2 = 9.14, p = 0.01) and practice (X2 = 4.71, p = 0.03) of generic substitution. Conclusions Our study found that pharmacy professionals working in community drug retail outlets in Jimma town had lack of knowledge about generic substitution. Conversely, an enormous amount of participants had positive attitude toward generic substitution and nearly half of them had practiced generic substitution. The year of experience had a significant effect on knowledge and practice of generic substitution.