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High levels of heterozygosity present a unique genome assembly challenge and can adversely impact downstream analyses, yet is common in sequencing datasets obtained from non-model organisms. Here we show that by re-assembling a heterozygous dataset with variant parameters and different assembly algorithms, we are able to generate assemblies whose protein annotations are statistically enriched for specific gene ontology categories. While total assembly length was not significantly affected by assembly methodologies tested, the assemblies generated varied widely in fragmentation level and we show local assembly collapse or expansion underlying the enrichment or depletion of specific protein functional groups. We show that these statistically significant deviations in gene ontology groups can occur in seemingly high-quality assemblies, and result from difficult-to-detect local sequence expansion or contractions. Given the unpredictable interplay between assembly algorithm, parameter, and biological sequence data heterozygosity, we highlight the need for better measures of assembly quality than N50 value, including methods for assessing local expansion and collapse. Author summary: In the genomic era, genomes must be reconstructed from fragments using computational methods, or assemblers. How do we know that a new genome assembly is correct? This is important because errors in assembly can lead to downstream problems in gene predictions and these inaccurate results can contaminate databases, affecting later comparative studies. A particular challenge occurs when a diploid organism inherits two highly divergent genome copies from its parents. While it is widely appreciated that this type of data is difficult for assemblers to handle properly, here we show that the process is prone to more errors than previously appreciated. Specifically, we document examples of regional expansion and collapse, affecting downstream gene prediction accuracy, but without changing the overall genome assembly size or other metrics of accuracy. Our results suggest that assembly evaluation methods should be altered to identify whether regional expansions and collapses are present in the genome assembly. [ABSTRACT FROM AUTHOR]