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INTRODUCTION Genetic interactions occur when mutations in different genes combine to result in a phenotype that is different from expectation based on those of the individual mutations. Negative genetic interactions occur when a combination of mutations leads to a fitness defect that is more exacerbated than expected. For example, synthetic lethality occurs when two mutations, neither of which is lethal on its own, generate an inviable double mutant. Alternatively, positive genetic interactions occur when genetic perturbations combine to generate a double mutant with a greater fitness than expected. Global digenic interaction studies have been useful for understanding the functional wiring diagram of the cell and may also provide insight into the genotype-to-phenotype relationship, which is important for tracking the missing heritability of human health and disease. Here we describe a network of higher-order trigenic interactions and explore its implications. RATIONALE Variation in phenotypic outcomes in different individuals is caused by genetic determinants that act as modifiers. Modifier loci are prevalent in human populations, but knowledge regarding how variants interact to modulate phenotype in different individuals is lacking. Similarly, in yeast, traits including conditional essentiality—in which certain genes are essential in one genetic background but nonessential in another—often result from an interplay of multiple modifier loci. Because complex modifiers may underlie the genetic basis of physiological states found in natural populations, it is critical to understand the landscape of higher-order genetic interactions. RESULTS To survey trigenic interactions, we designed query strains that sampled key features of the global digenic interaction network: (i) digenic interaction strength, (ii) average number of digenic interactions, and (iii) digenic interaction profile similarity. In total, we tested ~400,000 double and ~200,000 triple mutants for fitness defects and identified ~9500 digenic and ~3200 trigenic negative interactions. Although trigenic interactions tend to be weaker than digenic interactions, they were both enriched for functional relationships. About one-third of trigenic interactions identified “novel” connections that were not observed in our digenic control network, whereas the remaining approximately two-thirds of trigenic interactions “modified” a digenic interaction, suggesting that the global digenic interaction network is important for understanding the trigenic interaction network. Despite their functional enrichment, trigenic interactions also bridged distant bioprocesses. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance. CONCLUSION The extensive network of trigenic interactions and their ability to generate functionally diverse phenotypes suggest that higher-order genetic interactions may play a key role in the genotype-to-phenotype relationship, genome size, and speciation.