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Urbanization results in habitat loss and habitat fragmentation concurrently, both influencing biodiversity and ecological processes. To evaluate these impacts, it is important to understand the relationships between habitat loss and habitat fragmentation per se (HLHF) during urbanization. The objectives of this study were two-fold: 1) to quantify the different forms of the HLHF relationship during urbanization using multiple landscape metrics, and 2) to test the validity of the HLHF relations reported in the literature. Our analysis was based on a long-term urbanization dataset (1800–2000) of 16 large cities from around the world. Habitat area was represented as the percentage of non-built-up area in the landscape, while habitat fragmentation was measured using several landscape metrics. Our results show that the relationship between habitat loss and habitat fragmentation during urbanization is commonly monotonic—linear, exponential, or logarithmic, indicating that the degree of habitat fragmentation per se increases with habitat loss in general. We compared our results with 14 hypothesized HLHF relationships based on simulated landscapes found in the literature, and found that four of them were consistent with those of urbanization, whereas the other ten were not. Also, we identified six new HLHF relationships when fragmentation was measured by total core area, normalized total core area, patch density, edge density and landscape shape index, respectively. In addition, our study demonstrated that the “space-for-time” approach, frequently used in ecology and geography, generated specious HLHF relationships, suggesting that this approach …