Inter-organizational innovation is becoming an attractive development form in view of the complexity of many of today’s innovations. However, often inter-organizational innovation does not lead to the desired results. To understand this paradoxical situation this article of an inter-organizational R&D setting observes the emergence and evolution of knowledge boundaries and their relation to practice. Through a longitudinal exploratory field study we identify various boundary types that permeate with varying intensities the entire innovation process and through a comparison with the activities carried out throughout an R&D project we derive three theoretical explanations to the emergence of boundaries in inter-organizational R&D: sensemaking, strategizing and culture. As these boundaries have both positive and negative impact on collaboration, recommendations for the management of the boundaries – instead of their elimination – are provided in the conclusions.